Hydrazinyl-pyrrolo compounds and methods for producing a conjugate

ABSTRACT

The present disclosure provides conjugate structures and hydrazinyl-pyrrolo compound structures used to produce these conjugates. The disclosure also encompasses methods of production of such conjugates, as well as methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/555,283, filed Nov. 26, 2014, which claims priority benefit pusuantto 35 U.S.C. §119(e) of U.S. Provisional Application Ser. No.61/909,897, filed Nov. 27, 2013, the disclosures of which applicationsare incorporated herein by reference in their entirety.

INTRODUCTION

The field of protein-small molecule therapeutic conjugates has advancedgreatly, providing a number of clinically beneficial drugs with thepromise of providing more in the years to come. Protein-conjugatetherapeutics can provide several advantages, due to, for example,specificity, multiplicity of functions and relatively low off-targetactivity, resulting in fewer side effects. Chemical modification ofproteins may extend these advantages by rendering them more potent,stable, or multimodal.

A number of standard chemical transformations are commonly used tocreate and manipulate post-translational modifications on proteins.There are a number of methods where one is able to modify the sidechains of certain amino acids selectively. For example, carboxylic acidside chains (aspartate and glutamate) may be targeted by initialactivation with a water-soluble carbodiimide reagent and subsequentreaction with an amine Similarly, lysine can be targeted through the useof activated esters or isothiocyanates, and cysteine thiols can betargeted with maleimides and a-halo-carbonyls.

One significant obstacle to the creation of a chemically altered proteintherapeutic or reagent is the production of the protein in abiologically active, homogenous form. Conjugation of a drug ordetectable label to a polypeptide can be difficult to control, resultingin a heterogeneous mixture of conjugates that differ in the number ofdrug molecules attached and in the position of chemical conjugation. Insome instances, it may be desirable to control the site of conjugationand/or the drug or detectable label conjugated to the polypeptide usingthe tools of synthetic organic chemistry to direct the precise andselective formation of chemical bonds on a polypeptide.

SUMMARY

The present disclosure provides conjugate structures andhydrazinyl-pyrrolo compound structures used to produce these conjugates.The disclosure also encompasses methods of production of suchconjugates, as well as methods of using the same.

Aspects of the present disclosure include a conjugate comprising atleast one modified amino acid residue of formula (I):

wherein

m is 0 or 1;

R¹ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, and substituted heterocyclyl;

R² and R³ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, orR² and R³ are optionally cyclically linked to form a 5 or 6-memberedheterocyclyl;

Z¹, Z², Z³ and Z⁴ are each independently selected from CR¹¹, NR¹², O andS, wherein one of Z¹, Z², Z³ and Z⁴ is optional;

X⁵ is C;

Y⁵, R¹¹ and R¹² are each independently selected from hydrogen, halogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl,alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substitutedthioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl;

Q¹ is a bond to either Z⁴ or X⁵, wherein if Q¹ is a bond to Z⁴, then Z⁴is CR¹¹ or NR¹² and R¹¹ or R¹² is absent, or if Q¹ is a bond to X⁵, thenY⁵ is absent;

R¹⁵ is -L-W¹ or -L-W¹ is attached to one of Z¹, Z², Z³ or Z⁴, wherein if-L-W¹ is attached to one of Z¹, Z², Z³ or Z⁴, then R¹⁵ is selected fromhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl;

L is a linker comprising-(T¹-V¹)_(a)-(T²-V²)_(b)-(T³-V³),-(T⁴-V⁴)_(d)-(T⁵-V⁵), wherein a, b, c,d and e are each independently 0 or 1, where the sum of a, b, c, d and eis 1 to 5;

T¹, T², T³, T⁴ and T⁵ are each independently selected from(C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, (EDA)_(w), (PEG)_(n),(AA)_(p), —(CR¹³OH)_(h)—, piperidin-4-amino (P4A),para-amino-benzyloxycarbonyl (PABC), meta-amino-benzyloxycarbonyl(MABC), para-amino-benzyloxy (PABO), meta-amino-benzyloxy (MABO),para-aminobenzyl, an acetal group, a disulfide, a hydrazine, aprotease-cleavable moiety, a glucuronidase cleavable moiety, abeta-lactamase cleavable moiety, and an ester, wherein EDA is anethylene diamine moiety, PEG is a polyethylene glycol or a modifiedpolyethylene glycol, and AA is an amino acid residue;

w is an integer from 1 to 20;

n is an integer from 1 to 30;

p is an integer from 1 to 20;

h is an integer from 1 to 12; and

V¹, V², V³, V⁴ and V⁵ are each independently selected from the groupconsisting of a covalent bond, —CO—, —NR¹¹—, —CONR¹¹—, —NR¹¹CO—,—C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO₂—, —SO₂NR¹¹—, —NR¹¹SO₂— and—P(O)OH—,

wherein one of W¹ and W² is a polypeptide and the other is a chemicalentity, and

wherein when the sum of a, b, c, d and e is 2 and one of T¹-V¹, T²-V²,T³-V³, T⁴-V⁴ or T⁵-V⁵-V is (PEG)-CO, then n is not 6.

In some embodiments, the conjugate includes at least one modified aminoacid residue of formula (II):

wherein

m, R¹, R², R³′ X⁵, L, Q¹, W¹, W², Y⁵, Z¹, Z², Z³ and Z⁴ are as definedin formula (I).

In some embodiments, the conjugate includes at least one modified aminoacid residue of formula (III):

wherein

m, R¹, R², R³′ X⁵, L, W¹ and W² are as defined in formula (I);

X¹, X², X³ and X⁴ are each independently selected from C, N, O and S;

Y¹, Y², Y³, Y⁴ and Y⁵, if present, are each independently selected fromhydrogen, halogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, amino acyl, alkylamide, substituted alkylamide, sulfonyl,thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, wherein Y¹ and Y², Y² andY³, or Y³ and Y⁴ are optionally cyclically linked; and

Q¹ is a bond to either X⁴ or X⁵, wherein if Q¹ is a bond to X⁴, then Y⁴is absent, or if Q¹ is a bond to X⁵, then Y⁵ is absent.

In some embodiments, Y¹ and Y², Y² and Y³, or Y³ and Y⁴ are cyclicallylinked to form a fused benzo ring.

In some embodiments, the conjugate includes at least one modified aminoacid residue of formula (IVa) or (IVb):

wherein:

m, R¹, R², R³, R¹²′ X⁵, Y⁵, L, W¹ and W² are as defined in formula (I);X¹ and X³ are each independently O, S or NR¹²;

Y¹, Y², Y³ and Y⁴, if present, are each independently selected fromhydrogen, halogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, amino acyl, alkylamide, substituted alkylamide, sulfonyl,thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, wherein Y¹ and Y² or Y³ andY⁴ are optionally cyclically linked;

Q¹ is a bond to either X⁴ or X⁵, wherein if Q¹ is a bond to X⁴, then X⁴is C and Y⁴ is absent, or if Q¹ is a bond to X⁵, then Y⁵ is absent; and

Q² is a bond to either X³ or X⁵, wherein if Q² is a bond to X³, then X³is NR¹² and R¹² is absent, or if Q² is a bond to X⁵, then Y⁵ is absent.

In some embodiments, Y¹ and Y² or Y³ and Y⁴ are cyclically linked toform a fused benzo ring.

In some embodiments,

T¹ is selected from a (C₁-C₁₂)alkyl and a substituted (C₁-C₁₂)alkyl;

T², T³, T⁴ and T⁵ are each independently selected from (EDA)_(w),(PEG)_(n), (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, (AA),—(CR¹³OH)_(h)—, piperidin-4-amino, MABC, MABO, PABO, PABC,para-aminobenzyl, an acetal group, a disulfide, a hydrazine, aprotease-cleavable moiety, a glucuronidase cleavable moiety, abeta-lactamase cleavable moiety, an ester, (AA)_(p)-MABC-(AA)_(p),(AA)_(p)-MABO-(AA)_(p), (AA)_(p)-PABO-(AA)_(p) and (AA)_(p)-PABC-(AA);and

V¹, V², V³, V⁴ and V⁵ are each independently selected from the groupconsisting of: a covalent bond, —CO—, —NR¹¹—, —CONR¹¹—, —NR¹¹CO—,—C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO₂—, —SO₂NR¹¹—, —NR¹¹SO₂—, and—P(O)OH—;

wherein:

(PEG)_(n) is

where n is an integer from 1 to 30;

EDA is an ethylene diamine moiety having the following structure:

where q is an integer from 1 to 6 and r is 0 or 1; piperidin-4-amino is

each R¹¹ and R¹² is independently selected from hydrogen, an alkyl, asubstituted alkyl, a PEG, an aryl and a substituted aryl, wherein anytwo adjacent R¹² groups may be cyclically linked to form a piperazinylring; and

R¹³ is selected from hydrogen, an alkyl, a substituted alkyl, an aryl,and a substituted aryl.

In some embodiments, a, b, c and d are each 1; and e is 0.

In some embodiments, a, b and c are each 1; and d and e are each 0.

In some embodiments, a and b are each 1; and c, d and e are each 0.

In some embodiments, T¹, T², T³,T⁴ and T⁵ and V¹, V², V³,V⁴ and V⁵ areselected from the following table:

T¹ V¹ T² V² T³ V³ T⁴ V⁴ T⁵ V⁵ (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— — —— — — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— — — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) — — — — — — — (C₁-C₁₂)alkyl —CONR¹¹—(PEG)_(n) NR¹¹— — — — — — — (C₁-C₁₂)alkyl —CO— (AA)_(p) NR¹¹— (PEG)_(n)—NR¹¹— — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— — — — — — —(C₁-C₁₂)alkyl —CONR¹¹— (C₁-C₁₂)alkyl —NR¹¹— — — — — — — (C₁-C₁₂)alkyl—CONR^(11—) (PEG)_(n) —CO— (EDA)_(w) — — — — — (C₁-C₁₂)alkyl —CO—(EDA)_(w) — — — — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— (CR¹³OH)_(h)—CONR¹¹— (C₁-C₁₂)alkyl —CO— — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹—(C₁-C₁₂)alkyl —CO— — — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO—(AA)_(p) — — — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — MABO— — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABO — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABC — — —(C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— (CR¹³OH)_(h) —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (C₁-C₁₂)alkyl —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (C₁-C₁₂)alkyl —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p)- — — — PABC-(AA)_(p) (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p)- —PABC- — (AA)_(p) (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO—(AA)_(p)- — — — PABO — — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n)—CO— (AA)_(p) — PABO (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —SO₂—(AA)_(p) — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABC- —— — (AA)_(p) (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABC —(AA)_(p) — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— (CR¹³OH)_(h) —CONR¹¹—(PEG)_(n) —CO— — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO—MABC- — — — (AA)_(p) (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO—MABC — (AA)_(p) — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— MABO — — — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— MABO — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— PABO — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— PABC — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— MABC — (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (CR¹³OH)_(h) —CO— — — — — — — (C₁-C₁₂)alkyl —CONR¹¹—substituted —NR¹¹— (PEG)_(n) —CO— — — — — (C₁-C₁₂)alkyl (C₁-C₁₂)alkyl—SO₂— (C₁-C₁₂)alkyl —CO— — — — — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n)—CO— (AA)_(p) — PABC —NR¹¹— — — (C₁-C₁₂)alkyl —CONR¹¹— (C₁-C₁₂)alkyl —(CR¹³OH)_(h) —CONR¹¹— — — — — (C₁-C₁₂)alkyl —CO— P4A —CO— (C₁-C₁₂)alkyl—CO— (AA)_(p) — PABO —CO— (C₁-C₁₂)alkyl —CO— P4A —CO— (C₁-C₁₂)alkyl —CO—(AA)_(p) — PABO — (C₁-C₁₂)alkyl —CO— P4A —CO— (C₁-C₁₂)alkyl —CO—(AA)_(p) — PABC- — (AA)_(p) (C₁-C₁₂)alkyl —CO— P4A —CO— (C₁-C₁₂)alkyl—CO— (AA)_(p) — — —

In some embodiments, L is described by one of the following structures:

wherein:

each f is independently 0 or an integer from 1 to 12;

each w is independently 0 or an integer from 1 to 20;

each n is independently 0 or an integer from 1 to 30;

each p is independently 0 or an integer from 1 to 20;

each h is independently 0 or an integer from 1 to 12;

each R is independently hydrogen, alkyl, substituted alkyl, apolyethylene glycol moiety, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl,alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substitutedthioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl; and

each R′ is independently H, a sidechain group of an amino acid, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

In some embodiments, Q¹ is a bond to X⁴ and Y⁴ is absent.

In some embodiments, Q¹ is a bond to X⁵ and Y⁵ is absent.

In some embodiments, m is 1.

In some embodiments, R² and R³ are each independently selected fromalkyl and substituted alkyl.

In some embodiments, R² and R³ are each methyl.

In some embodiments, X¹, X², X³ and X⁴ are each C.

In some embodiments, Y¹, Y² and Y³ are each H, and one of either Y⁴ orY⁵ is H.

In some embodiments, the conjugate includes at least one modified aminoacid residue of formula (IIIa):

In some embodiments, the conjugate includes at least one modified aminoacid residue of formula (IIIb):

In some embodiments, the conjugate includes at least one modified aminoacid residue of formula (IIId):

In some embodiments, the conjugate comprises at least one modified aminoacid residue of formula (IIIe):

In some embodiments, the chemical entity is a drug or a detectablelabel.

In some embodiments, W¹ is the chemical entity, and W² is thepolypeptide.

In some embodiments, W¹ is the polypeptide, and W² is the chemicalentity.

Aspects of the present disclosure includes a compound of formula (V):

wherein

one of Q³ and Q⁴ is (CH₂)_(m)NR³NHR² and the other is Y⁴;

m is 0 or 1;

R² and R³ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocycly, or R²and R³ are optionally cyclically linked to form a 5 or 6-memberedheterocyclyl 1;

X¹, X², X³ and X⁴ are each independently selected from C, N, O and S,wherein one of X¹, X², X³ and X⁴ is optional;

Y¹, Y², Y³ and Y⁴ are each independently selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl, wherein Y¹ and Y² or Y² and Y³ are optionallycyclically linked, and wherein when Q⁴ is Y⁴, then Y³ and Y⁴ areoptionally cyclically linked;

one of R¹⁶, Y¹, Y², Y¹ or Q⁴ is L-W¹, wherein if Q⁴ is L-W¹, then Q³ is—(CH₂)_(m)NR³NHR² and Y⁴ is absent; and wherein if one of Y¹, Y², Y³ orQ⁴ is L-W¹, then R¹⁶ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

L is a linker comprising-(T¹-V¹)_(a)-(T²-V²)_(b)-(T³-V³)_(c)-(T⁴-V⁴)_(d)-(T⁵-V⁵)_(e)-, whereina, b, c, d and e are each independently 0 or 1, where the sum of a, b,c, d and e is 1 to 5;

T¹, T², T³, T⁴ and T⁵ are each independently selected from(C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, (EDA)_(w), (PEG)_(n),(AA)_(p), —(CR¹³OH)_(h)—, piperidin-4-amino (P4A),para-amino-benzyloxycarbonyl (PABC), a meta-amino-benzyloxycarbonyl(MABC), a para-amino-benzyloxy (PABO), a meta-amino-benzyloxy (MABO),para-aminobenzyl, an acetal group, a disulfide, a hydrazine, aprotease-cleavable moiety, a glucuronidase cleavable moiety, abeta-lactamase cleavable moiety, and an ester, wherein EDA is anethylene diamine moiety, PEG is a polyethylene glycol or a modifiedpolyethylene glycol, and AA is an amino acid residue;

w is an integer from 1 to 20;

n is an integer from 1 to 30;

p is an integer from 1 to 20;

h is an integer from 1 to 12;

V¹, V², V³, V⁴ and V⁵ are each independently selected from the groupconsisting of a covalent bond, —CO—, —NR¹¹—, —CONR¹¹—, —NR¹¹CO—,—C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO₂—, —SO₂NR¹¹—, —NR¹¹SO₂— and—P(O)OH—; and

W¹ is selected from a polypeptide and a chemical entity, and

wherein when the sum of a, b, c, d and e is 2 and one of T¹-V¹, T²-V²,T³-V³, T⁴-V⁴ or T⁵-V⁵ is (PEG)_(n)-CO, then n is not 6.

In some embodiments, the compound is a compound of formula (VI):

wherein

Q³, Q⁴, X¹, X², X³, X⁴, L, W¹, Y¹, Y², and Y³ are as defined in formula(V).

In some embodiments, the compound is a compound of formula (VIIa) or(VIIb):

wherein:

X¹ and X³ are each independently O, S or NR¹²;

in formula (VIIa), one of Q³ and Q⁴ is —(CH₂)_(m)NR³NHR² and the otheris Y⁴;

in formula (VIIb), one of Q³ and R¹², if present, is —(CH₂)NR³NHR² andthe other is Y³;

Y¹, Y², Y³ and Y⁴, if present, are each independently selected fromhydrogen, halogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, amino acyl, alkylamide, substituted alkylamide, sulfonyl,thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, wherein Y¹ and Y² areoptionally cyclically linked, and wherein when Q⁴ is Y⁴, then Y³ and Y⁴are optionally cyclically linked; and

m, R² and R³ are as defined in formula (V).

In some embodiments, Y¹ and Y² or Y³ and Y⁴ are cyclically linked toform a fused benzo ring.

In some embodiments,

T¹ is selected from a (C₁-C₁₂)alkyl and a substituted (C₁-C₁₂)alkyl;

T², T³ , T⁴ and T⁵ are each independently selected from (EDA)_(w),(PEG)_(n), (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, (AA)_(p),—(CR¹³OH)_(h)—, piperidin-4-amino, MABC, MABO, PABO, PABC,para-aminobenzyl, an acetal group, a disulfide, a hydrazine, aprotease-cleavable moiety, a glucuronidase cleavable moiety, abeta-lactamase cleavable moiety, an ester, (AA)_(p)-MABC-(AA)_(p),(AA)-MABO-(AA), (AA)_(p)-PABO-(AA)_(p) and (AA)_(p)-PABC-(AA); and

V¹, V², V³, V⁴ and V⁵ are each independently selected from the groupconsisting of: a covalent bond, —CO—, —NR¹¹—, —CONR¹¹—, —NR¹¹CO—,—C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO₂—, —SO₂NR¹¹—, —NR¹¹SO₂—, and—P(O)OH—;

wherein:

(PEG) is

where n is an integer from 1 to 30;

EDA is an ethylene diamine moiety having the following structure:

where q is an integer from 1 to 6 and r is 0 or 1; piperidin-4-amino is

each R¹¹ and R¹² is independently selected from hydrogen, an alkyl, asubstituted alkyl, a polyethylene glycol moiety, an aryl and asubstituted aryl, wherein any two adjacent R¹² groups may be cyclicallylinked to form a piperazinyl ring; and

R¹³ is selected from hydrogen, an alkyl, a substituted alkyl, an aryl,and a substituted aryl.

In some embodiments, a, b, c and d are each 1; and e is 0.

In some embodiments, a, b and c are each 1; and d and e are 0.

In some embodiments, a and b are each 1; and c, d and e are 0.

In some embodiments, T¹, T², T³, T⁴ and T⁵ and V¹, V², V³, V⁴ and V⁵ areselected from the following table:

T¹ V¹ T² V² T³ V³ T⁴ V⁴ T⁵ V⁵ (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— — —— — — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— — — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) — — — — — — — (C₁-C₁₂)alkyl —CONR¹¹—(PEG)_(n) —NR¹¹— — — — — — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹—(PEG)_(n) —NR¹¹— — — — — (C₁-C12)alkyl —CO— (EDA)_(w) —CO— — — — — — —(C₁-C₁₂)alkyl —CONR¹¹— (C₁-C₁₂)alkyl —NR¹¹— — — — — — — (C₁-C₁₂)alkyl—CONR¹¹— (PEG)_(n) —CO— (EDA)_(w) — — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w)— — — — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— (CR¹³OH)_(h) —CONR¹¹—(C₁-C₁₂)alkyl —CO— — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (C₁-C₁₂)alkyl—CO— — — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — — — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — MABO — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABO — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABC — — —(C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— (CR¹³OH)_(h) —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (C₁-C₁₂)alkyl —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (C₁-C₁₂)alkyl —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p)- — — — PABC-(AA)_(p) (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p)- —PABC- — (AA)_(p) (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO—(AA)_(p)- — — — PABO (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO—(AA)_(p) — PABO — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —SO₂—(AA)_(p) — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) PABC- — —— (AA)_(p) (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) PABC —(AA)_(p) — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— (CR¹³OH)_(h) —CONR¹¹—(PEG)_(n) —CO— — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO—MABC- — — — (AA)_(p) (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO—MABC — (AA)_(p) — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— MABO — — — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— MABO — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— PABO — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— PABC — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— MABC — (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (CR¹³OH)_(h) —CO— — — — — — — (C₁-C₁₂)alkyl —CONR¹¹—substituted —NR¹¹— (PEG)_(n) —CO— — — — — (C₁-C₁₂)alkyl (C₁-C₁₂)alkyl—SO₂— (C₁-C₁₂)alkyl —CO— — — — — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n)—CO— (AA)_(p) — PABC —NR¹¹— — — (C₁-C₁₂)alkyl —CONR¹¹— (C₁-C₁₂)alkyl —(CR¹³OH)_(h) —CONR¹¹— — — — — (C₁-C₁₂)alkyl —CO— P4A —CO— (C₁-C₁₂)alkyl—CO— (AA)_(p) — PABO —CO— (C₁-C₁₂)alkyl —CO— P4A —CO— (C₁-C₁₂)alkyl —CO—(AA)_(p) — PABO — (C₁-C₁₂)alkyl —CO— P4A —CO— (C₁-C₁₂)alkyl —CO—(AA)_(p) — PABC- — (AA)_(p) (C₁-C₁₂)alkyl —CO— P4A —CO— (C₁-C₁₂)alkyl—CO— (AA)_(p) — — —

In some embodiments, L is described by one of the following structures:

wherein:

each f is independently 0 or an integer from 1 to 12;

each w is independently 0 or an integer from 1 to 20;

each n is independently 0 or an integer from 1 to 30;

each p is independently 0 or an integer from 1 to 20;

each h is independently 0 or an integer from 1 to 12;

each R is independently hydrogen, alkyl, substituted alkyl, apolyethylene glycol moiety, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl,alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substitutedthioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl; and

each R′ is independently H, a sidechain group of an amino acid, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

In some embodiments, Q³ is —(CH₂)_(m)NR³NHR² and Q⁴ is Y⁴.

In some embodiments, Q⁴ is —(CH₂)_(m)NR³NHR² and Q³ is Y⁴.

In some embodiments, m is 1.

In some embodiments, R² and R³ are each independently selected fromalkyl and substituted alkyl.

In some embodiments, R² and R³ are each methyl.

In some embodiments, X¹, X², X³ and X⁴ are each C.

In some embodiments, Y¹, Y², Y³ and Y⁴ are each H.

In some embodiments, X¹ and X³ are each S and X⁴ is C.

In some embodiments, X¹ and X³ are each O and X⁴ is C.

In some embodiments, X¹ and X³ are each NR¹² and X⁴ is C.

In some embodiments, the compound is a compound of formula (VIII):

In some embodiments, the compound is a compound of formula (VIIIa):

In some embodiments, the compound is a compound of formula (IX):

In some embodiments, the compound is a compound of formula (IXa):

In some embodiments, W¹ is a drug or a detectable label.

In some embodiments, the detectable label comprises a fluorophore.

Aspects of the present disclosure include a method of producing apolypeptide conjugate. The method includes combining in a reactionmixture: a compound as described herein and a second compound comprisinga reactive aldehyde group or a reactive ketone group, where thecombining is under reaction conditions suitable to promote reactionbetween the compound and the reactive aldehyde group or reactive ketonegroup of the second compound to form the conjugate, and isolating theconjugate from the reaction mixture.

In some embodiments, W¹ is the chemical entity, and the second compoundcomprises the polypeptide.

In some embodiments, W¹ is the polypeptide, and the second compoundcomprises the chemical entity.

In some embodiments, the reaction mixture has a pH of 7.

In some embodiments, the reaction conditions are at a temperature of 37°C.

Aspects of the present disclosure include a pharmaceutical composition.The pharmaceutical composition includes a conjugate as described hereinand a pharmaceutically acceptable excipient.

Aspects of the present disclosure include a method of delivering aconjugate to a subject. The method includes administering to the subjectan effective amount of a conjugate as described herein.

Aspects of the present disclosure include a method of treating acondition in a subject. The method includes administering to the subjecthaving the condition a therapeutically effective amount of apharmaceutical composition comprising a conjugate as described herein,where the administering is effective to treat the condition in thesubject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (panels A and B) show a reaction schemes for the production of apolypeptide conjugate that includes a hydrazinyl-pyrrolo couplingmoiety, according to embodiments of the present disclosure.

FIG. 2 (panel A) shows a reaction scheme for the synthesis of afunctionalized detectable label, according to embodiments of the presentdisclosure. FIG. 2 (panel B) shows a schematic of a conjugation reactionof the functionalized detectable label to an antibody, according toembodiments of the present disclosure.

FIG. 3 and FIG. 4 show reaction schemes for the synthesis of compoundHIPS Indole E (CO₂H) PEG₂ Maytansine, according to embodiments of thepresent disclosure, see e.g., Example 1.

FIG. 5 and FIG. 6 show reaction schemes for the synthesis of compoundHIPS Indole E (CO₂H) PEG₂ Maytansine according to embodiments of thepresent disclosure, see e.g., Example 2.

FIG. 7 and FIG. 8 show reaction schemes for the synthesis of compoundHIPS Indole N (CONH₂) PEG₂ Maytansine according to embodiments of thepresent disclosure, see e.g., Example 3.

FIG. 9 shows a reaction scheme for the synthesis of compound HIPS IndoleCadaverine Alexa Fluor 555 according to embodiments of the presentdisclosure, see e.g., Example 4.

FIG. 10 and FIG. 11 show reaction schemes for the synthesis of compoundHIPS Indole E (CO₂H) PEG₂ NH ATTO 550 according to embodiments of thepresent disclosure, see e.g., Example 5.

FIG. 12 and FIG. 13 show reaction schemes for the synthesis of compoundHIPS Indole C (SO₃H) PEG₂ NH ATTO 550 according to embodiments of thepresent disclosure, see e.g., Example 6.

FIGS. 14 to 19 show reaction schemes for the synthesis of compound HIPSIndole G PEG₆ Val Cit PABC NMC₃ Maytansine according to embodiments ofthe present disclosure, see e.g., Example 7 (where, as used herein, NMC₃represents the group —N(CH₃)—(CH₂)₃—).

FIG. 20 shows a reaction scheme for the synthesis of compound HIPSIndole C (SO₃H) Maytansine according to embodiments of the presentdisclosure, see e.g., Example 8.

FIG. 21 and FIG. 22 show reaction schemes for the synthesis of compoundHIPS Indole G PEG₆ Maytansine according to embodiments of the presentdisclosure, see e.g., Example 9.

FIG. 23 shows a reaction scheme for the synthesis of compound HIPSIndole N ((OH)₃AcNH-β-Glc) PEG₂ Maytansine according to embodiments ofthe present disclosure, see e.g., Example 10.

FIG. 24 shows a reaction scheme for the synthesis of compound HIPSIndole Ethylenediamine Tartaric Acid (OH)₂ Beta Alanine Maytansineaccording to embodiments of the present disclosure, see e.g., Example11.

FIG. 25 shows a reaction scheme for the synthesis of compound HIPSIndole Glycine Dihydroxypeptoid (OH)₂ Beta Alanine Maytansine accordingto embodiments of the present disclosure, see e.g., Example 12.

FIG. 26 shows a reaction scheme for the synthesis of compound HIPSIndole Glycine Trihydroxypeptoid (OH)₃ Beta Alanine Maytansine accordingto embodiments of the present disclosure, see e.g., Example 13.

FIG. 27 shows a reaction scheme for the synthesis of compound HIPSIndole Glycine Trimethoxypeptoid Beta Alanine Maytansine according toembodiments of the present disclosure, see e.g., Example 14.

FIG. 28 shows a reaction scheme for the synthesis of compound HIPSIndole Glycine₃ Maytansine according to embodiments of the presentdisclosure, see e.g., Example 15.

FIG. 29 and FIG. 30 show reaction schemes for the synthesis of compoundHIPS Indole Aminoethylglycine PEG₂ Maytansine according to embodimentsof the present disclosure, see e.g., Example 16.

FIG. 31 and FIG. 32 show reaction schemes for the synthesis of compoundHIPS Indole Ethylenediamine Thiourea PEG₂ Maytansine according toembodiments of the present disclosure, see e.g., Example 17.

FIGS. 33A-33C show a size exclusion chromatography (SEC) trace (FIG.33A), a hydrophobic interaction column (HIC) trace (FIG. 33B), and amass spectrometer (MS) trace (FIG. 33C) of an aldehyde-tagged antibodyconjugated to HIPS Serine PEG₂ Maytansine, according to embodiments ofthe present disclosure.

FIGS. 34A-34C show a size exclusion chromatography (SEC) trace (FIG.34A), a hydrophobic interaction column (HIC) trace (FIG. 34B), and amass spectrometer (MS) trace (FIG. 34C) of an aldehyde-tagged antibodyconjugated to HIPS Phosphoserine PEG₂ Maytansine, according toembodiments of the present disclosure.

FIGS. 35A-35C show a size exclusion chromatography (SEC) trace (FIG.35A), a hydrophobic interaction column (HIC) trace (FIG. 35B), and amass spectrometer (MS) trace (FIG. 35C) of an aldehyde-tagged antibodyconjugated to HIPS Cysteic Acid PEG₂ Maytansine, according toembodiments of the present disclosure.

FIGS. 36A-36C show a size exclusion chromatography (SEC) trace (FIG.36A), a hydrophobic interaction column (HIC) trace (FIG. 36B), and amass spectrometer (MS) trace (FIG. 36C) of an aldehyde-tagged antibodyconjugated to HIPS Glutamic Acid PEG₂ Maytansine, according toembodiments of the present disclosure.

FIGS. 37A-37B show a size exclusion chromatography (SEC) trace (FIG.37A) and a hydrophobic interaction column (HIC) trace (FIG. 37B) of analdehyde-tagged antibody conjugated to HIPS Asparagine PEG₂ Maytansine,according to embodiments of the present disclosure.

FIGS. 38A-38B show a size exclusion chromatography (SEC) trace (FIG.38A) and a hydrophobic interaction column (HIC) trace (FIG. 38B) of analdehyde-tagged antibody conjugated to HIPS Phosphotyrosine PEG₂Maytansine, according to embodiments of the present disclosure.

FIGS. 39A-39C show a size exclusion chromatography (SEC) trace (FIG.39A), a hydrophobic interaction column (HIC) trace (FIG. 39B), and amass spectrometer (MS) trace (FIG. 39C) of an aldehyde-tagged antibodyconjugated to AzaHIPS Glutamic Acid PEG₂ Maytansine, according toembodiments of the present disclosure.

FIGS. 40A-40C show a size exclusion chromatography (SEC) trace (FIG.40A), a hydrophobic interaction column (HIC) trace (FIG. 40B), and amass spectrometer (MS) trace (FIG. 40C) of an aldehyde-tagged antibodyconjugated to HIPS Tartaric Acid Maytansine, according to embodiments ofthe present disclosure.

FIGS. 41A-41C show a size exclusion chromatography (SEC) trace (FIG.41A), a hydrophobic interaction column (HIC) trace (FIG. 41B), and amass spectrometer (MS) trace (FIG. 41C) of an aldehyde-tagged antibodyconjugated to HIPS Dihydroxy Maytansine, according to embodiments of thepresent disclosure.

FIGS. 42A-42C show a size exclusion chromatography (SEC) trace (FIG.42A), a hydrophobic interaction column (HIC) trace (FIG. 42B), and amass spectrometer (MS) trace (FIG. 42C) of an aldehyde-tagged antibodyconjugated to HIPS Glutamic Acid C₃ Maytansine, according to embodimentsof the present disclosure.

FIGS. 43A-43C show a size exclusion chromatography (SEC) trace (FIG.43A), a hydrophobic interaction column (HIC) trace (FIG. 43B), and amass spectrometer (MS) trace (FIG. 43C) of an aldehyde-tagged antibodyconjugated to HIPS Trimethoxy Maytansine, according to embodiments ofthe present disclosure.

FIG. 44 shows a hydrophobic interaction column (HIC) trace of analdehyde-tagged antibody conjugated to HIPS Glc NAc PEG₂ Ac₃ Maytansine,according to embodiments of the present disclosure.

FIG. 45 shows a hydrophobic interaction column (HIC) trace of analdehyde-tagged antibody conjugated to HIPS Glc NAc PEG₂ Maytansine,according to embodiments of the present disclosure.

FIGS. 46A-46B show a hydrophobic interaction column (HIC) trace (FIG.46A) and a mass spectrometer (MS) trace (FIG. 46B) of an aldehyde-taggedantibody conjugated to HIPS Nit PEG₂ Maytansine, according toembodiments of the present disclosure.

FIGS. 47A-47B show a hydrophobic interaction column (HIC) trace (FIG.47A) and a mass spectrometer (MS) trace (FIG. 47B) of an aldehyde-taggedantibody conjugated to HIPS PEG₂ MMAF, according to embodiments of thepresent disclosure.

FIG. 48 shows a hydrophobic interaction column (HIC) trace of analdehyde-tagged antibody conjugated to HIPS S PEG₄ Maytansine, accordingto embodiments of the present disclosure.

FIG. 49 shows a hydrophobic interaction column (HIC) trace of analdehyde-tagged antibody conjugated to HIPS S PEG₆ Maytansine, accordingto embodiments of the present disclosure.

FIG. 50 shows a hydrophobic interaction column (HIC) trace of analdehyde-tagged antibody conjugated to HIPS-S-C₅-Maytansine, accordingto embodiments of the present disclosure.

FIG. 51 shows a hydrophobic interaction column (HIC) trace of analdehyde-tagged antibody conjugated to HIPS G PEG₆ Maytansine, accordingto embodiments of the present disclosure.

FIG. 52 shows a hydrophobic interaction column (HIC) trace of analdehyde-tagged antibody conjugated to HIPS PEG₆ Val Cit PABC NMC₃Maytansine, according to embodiments of the present disclosure.

FIG. 53 shows a hydrophobic interaction column (HIC) trace of analdehyde-tagged antibody conjugated to HIPS Gly PEG₆ Val Cit PABC MMAE,according to embodiments of the present disclosure.

FIG. 54 shows a hydrophobic interaction column (HIC) trace of analdehyde-tagged antibody conjugated to HIPS Cysteic Acid Maytansine,according to embodiments of the present disclosure.

FIG. 55 shows images of SDS-PAGE gels showing an aldehyde-taggedantibody conjugated to HIPS Indole E (CO₂H) PEG₂ NH Alexa Fluor 488,according to embodiments of the present disclosure.

FIG. 56 shows a hydrophobic interaction column (HIC) trace of analdehyde-tagged antibody conjugated to HIPS PEG₆ Maytansine, accordingto embodiments of the present disclosure.

FIG. 57 shows a hydrophobic interaction column (HIC) trace of analdehyde-tagged antibody conjugated to PIPS PEG₂ Maytansine, accordingto embodiments of the present disclosure.

FIG. 58 shows a hydrophobic interaction column (HIC) trace of analdehyde-tagged antibody conjugated to HIPS Trihydroxy Maytansine,according to embodiments of the present disclosure.

FIG. 59 shows a hydrophobic interaction column (HIC) trace of analdehyde-tagged antibody conjugated to HIPS Lysine PEG₂ Maytansine,according to embodiments of the present disclosure.

FIG. 60 shows a reaction scheme for the synthesis of(25)-8-(1-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-1-(((1⁴S,1⁶S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl)oxy)-2,3-dimethyl-1,4,7-trioxo-11,14-dioxa-3,8-diazaheptadecan-17-oicacid (Fmoc-HIPS-PAPip(PEG2(CO2H))-maytansine) according to embodimentsof the present disclosure, see e.g., Example 20.

FIG. 61 shows a reaction scheme for the synthesis of(2S,5S,18S)-1-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)amino)-18-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-5-isopropyl-2-methyl-1,4,7,17-tetraoxo-10,13-dioxa-3,6,16-triazahenicosan-21-oicacid (HIPS-Glu(OH)-PEG2-Val-Ala-PABC-MMAD) according to embodiments ofthe present disclosure, see e.g., Example 21.

FIG. 62 shows a reaction scheme for the synthesis of13-(1-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-2,2-dimethyl-4,14-dioxo-3,7,10-trioxa-13-azaheptadecan-17-oicacid (Fmoc-HIPS-PAPip(PEG2(CO2t-Bu))CO2H) according to embodiments ofthe present disclosure, see e.g., Example 22.

FIG. 63 shows a reaction scheme for the synthesis of(2S,5S)-1-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)amino)-11-(1-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-5-isopropyl-2-methyl-1,4,7,10-tetraoxo-14,17-dioxa-3,6,11-triazaicosan-20-oicacid according to embodiments of the present disclosure, see e.g.,Example 23.

FIG. 64 shows a reaction scheme for the synthesis of(6S,9S)-1-amino-6-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-15-(1-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-9-isopropyl-1,8,11,14-tetraoxo-18,21-dioxa-2,7,10,15-tetraazatetracosan-24-oicacid according to embodiments of the present disclosure, see e.g.,Example 24.

FIG. 65 shows a reaction scheme for the synthesis of(6S,9S)-1-amino-6-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-15-(1-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanoyl)piperidin-4-yl)-9-isopropyl-1,8,11,14-tetraoxo-18,21-dioxa-2,7,10,15-tetraazatetracosan-24-oicacid according to embodiments of the present disclosure, see e.g.,Example 25.

FIGS. 66A-66C show a size exclusion chromatography (SEC) trace (FIG.66A), a hydrophobic interaction column (HIC) trace (FIG. 66B), and amass spectrometer (MS) trace (FIG. 66C) of an aldehyde-tagged antibodyconjugated to HIPS-PAPip(PEG2(CO2H))-Maytansine, according toembodiments of the present disclosure.

FIG. 67 shows a hydrophobic interaction column (HIC) trace of analdehyde-tagged antibody conjugated to HIPS-GlutamicAcid-PEG2-Valine-Alanine-PABC-Maytansine, according to embodiments ofthe present disclosure.

FIG. 68 shows a hydrophobic interaction column (HIC) trace of analdehyde-tagged antibody conjugated toHIPS-PAPip(PEG2(CO2H))-Valine-Alanine-PABC-MMAD, according toembodiments of the present disclosure.

FIGS. 69A-69B show a size exclusion chromatography (SEC) trace (FIG.69A) and a hydrophobic interaction column (HIC) trace (FIG. 69B) of analdehyde-tagged antibody conjugated toHIPS-PAPip(PEG2(CO2H))-Valine-Citrulline-PABC-MMAD, according toembodiments of the present disclosure.

FIGS. 70A-70B show a size exclusion chromatography (SEC) trace (FIG.70A) and a hydrophobic interaction column (HIC) trace (FIG. 70B) of analdehyde-tagged antibody conjugated toAzaHIPS-PAPip(PEG2(CO2H))-Valine-Citrulline-PABC-MMAD, according toembodiments of the present disclosure.

FIGS. 71A-71C show a size exclusion chromatography (SEC) trace (FIG.71A), a hydrophobic interaction column (HIC) trace (FIG. 71B), and amass spectrometer (MS) trace (FIG. 71C) of an aldehyde-tagged antibodyconjugated to HIPS-Glutamic Acid-PEG2-Valine-Citrulline-PABC-Maytansine,according to embodiments of the present disclosure.

FIGS. 72A-72C show a size exclusion chromatography (SEC) trace (FIG.72A), a hydrophobic interaction column (HIC) trace (FIG. 72B), and amass spectrometer (MS) trace (FIG. 72C) of an aldehyde-tagged antibodyconjugated to HIPS-Asparagine-PEG2-Maytansine, according to embodimentsof the present disclosure.

FIGS. 73A-73C show a size exclusion chromatography (SEC) trace (FIG.73A), a hydrophobic interaction column (HIC) trace (FIG. 73B), and amass spectrometer (MS) trace (FIG. 73C) of an aldehyde-tagged antibodyconjugated to HIPS-Alanine-PEG2-Maytansine, according to embodiments ofthe present disclosure.

DEFINITIONS

The following terms have the following meanings unless otherwiseindicated. Any undefined terms have their art recognized meanings.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groupshaving from 1 to 10 carbon atoms and such as 1 to 6 carbon atoms, or 1to 5, or 1 to 4, or 1 to 3 carbon atoms. This term includes, by way ofexample, linear and branched hydrocarbyl groups such as methyl (CH₃—),ethyl (CH₃CH₂—), n-propyl (CH₃CH₂CH₂—), isopropyl ((CH₃)₂CH—), n-butyl(CH₃CH₂CH₂CH₂—), isobutyl ((CH₃)₂CHCH₂—), sec-butyl ((CH₃)(CH₃CH₂)CH—),t-butyl ((CH₃)₃C—), n-pentyl (CH₃CH₂CH₂CH₂CH₂—), and neopentyl((CH₃)₃CCH₂—)

The term “substituted alkyl” refers to an alkyl group as defined hereinwherein one or more carbon atoms in the alkyl chain have been optionallyreplaced with a heteroatom such as —O—, —N—, —S—, —S(O)— (where n is 0to 2), —NR— (where R is hydrogen or alkyl) and having from 1 to 5substituents selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-aryl,—SO₂-heteroaryl, and —NR^(a)R^(b), wherein R′ and R″ may be the same ordifferent and are chosen from hydrogen, optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl andheterocyclic.

“Alkylene” refers to divalent aliphatic hydrocarbyl groups preferablyhaving from 1 to 6 and more preferably 1 to 3 carbon atoms that areeither straight-chained or branched, and which are optionallyinterrupted with one or more groups selected from —O—, —NR¹⁰—,—NR¹⁰C(O)—, —C(O)NR¹⁰— and the like. This term includes, by way ofexample, methylene (—CH₂—), ethylene (—CH₂CH₂—), n-propylene(—CH₂CH₂CH₂—), iso-propylene (—CH₂CH(CH₃)—), (—C(CH₃)₂CH₂CH₂—),(—C(CH₃)₂CH₂C(O)—), (—C(CH₃)₂CH₂C(O)NH—), (—CH(CH₃)CH₂—), and the like.

“Substituted alkylene” refers to an alkylene group having from 1 to 3hydrogens replaced with substituents as described for carbons in thedefinition of “substituted” below.

The term “alkane” refers to alkyl group and alkylene group, as definedherein.

The term “alkylaminoalkyl”, “alkylaminoalkenyl” and “alkylaminoalkynyl”refers to the groups R′NHR″— where R′ is alkyl group as defined hereinand R″ is alkylene, alkenylene or alkynylene group as defined herein.

The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl and-substituted alkylene-aryl where alkylene, substituted alkylene and arylare defined herein.

“Alkoxy” refers to the group —O-alkyl, wherein alkyl is as definedherein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. Theterm “alkoxy” also refers to the groups alkenyl-O—, cycloalkyl-O—,cycloalkenyl-O—, and alkynyl-O—, where alkenyl, cycloalkyl,cycloalkenyl, and alkynyl are as defined herein.

The term “substituted alkoxy” refers to the groups substituted alkyl-O—,substituted alkenyl-O—, substituted cycloalkyl-O—, substitutedcycloalkenyl-O—, and substituted alkynyl-O— where substituted alkyl,substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyland substituted alkynyl are as defined herein.

The term “alkoxyamino” refers to the group NH-alkoxy, wherein alkoxy isdefined herein.

The term “haloalkoxy” refers to the groups alkyl-O— wherein one or morehydrogen atoms on the alkyl group have been substituted with a halogroup and include, by way of examples, groups such as trifluoromethoxy,and the like.

The term “haloalkyl” refers to a substituted alkyl group as describedabove, wherein one or more hydrogen atoms on the alkyl group have beensubstituted with a halo group. Examples of such groups include, withoutlimitation, fluoroalkyl groups, such as trifluoromethyl, difluoromethyl,trifluoroethyl and the like.

The term “alkylalkoxy” refers to the groups -alkylene-O-alkyl,alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, andsubstituted alkylene-O-substituted alkyl wherein alkyl, substitutedalkyl, alkylene and substituted alkylene are as defined herein.

The term “alkylthioalkoxy” refers to the group -alkylene-S-alkyl,alkylene-S-substituted alkyl, substituted alkylene-S-alkyl andsubstituted alkylene-S-substituted alkyl wherein alkyl, substitutedalkyl, alkylene and substituted alkylene are as defined herein.

“Alkenyl” refers to straight chain or branched hydrocarbyl groups havingfrom 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and havingat least 1 and preferably from 1 to 2 sites of double bond unsaturation.This term includes, by way of example, bi-vinyl, allyl, andbut-3-en-1-yl. Included within this term are the cis and trans isomersor mixtures of these isomers.

The term “substituted alkenyl” refers to an alkenyl group as definedherein having from 1 to 5 substituents, or from 1 to 3 substituents,selected from alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

“Alkynyl” refers to straight or branched monovalent hydrocarbyl groupshaving from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms andhaving at least 1 and preferably from 1 to 2 sites of triple bondunsaturation. Examples of such alkynyl groups include acetylenyl(—C≡CH), and propargyl (—CH₂C≡CH).

The term “substituted alkynyl” refers to an alkynyl group as definedherein having from 1 to 5 substituents, or from 1 to 3 substituents,selected from alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl, and —SO₂-heteroaryl.

“Alkynyloxy” refers to the group —O-alkynyl, wherein alkynyl is asdefined herein. Alkynyloxy includes, by way of example, ethynyloxy,propynyloxy, and the like.

“Acyl” refers to the groups H-C(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—,substituted alkynyl-C(O)—, cycloalkyl-C(O)—, substitutedcycloalkyl-C(O)—, cycloalkenyl-C(O)—, substituted cycloalkenyl-C(O)—,aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—, substitutedheteroaryl-C(O)—, heterocyclyl-C(O)—, and substitutedheterocyclyl-C(O)—, wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein. For example, acylincludes the “acetyl” group CH₃C(O)—

“Acylamino” refers to the groups NR²⁰C(O)alkyl, —NR²⁰C(O)substitutedalkyl, N R²⁰C(O)cycloalkyl, —NR²⁰C(O)substituted cycloalkyl,—NR²⁰C(O)cycloalkenyl, —NR²⁰C(O)substituted cycloalkenyl,—NR²⁰C(O)alkenyl, —NR²⁰C(O)substituted alkenyl, —NR²⁰C(O)alkynyl,—NR²⁰C(O)substituted alkynyl, —NR²⁰C(O)aryl, —NR²⁰C(O)substituted aryl,—NR²⁰C(O)heteroaryl, —NR²⁰C(O)substituted heteroaryl,—NR²⁰C(O)heterocyclic, and —NR²⁰C(O)substituted heterocyclic, whereinR²⁰ is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Aminocarbonyl” or the term “aminoacyl” refers to the group—C(O)NR²¹R²², wherein R²¹ and R²² independently are selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic and where R²¹ and R²² are optionally joinedtogether with the nitrogen bound thereto to form a heterocyclic orsubstituted heterocyclic group, and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Aminocarbonylamino” refers to the group —NR²¹C(O)NR²²R²³ where R²¹,R²², and R²³ are independently selected from hydrogen, alkyl, aryl orcycloalkyl, or where two R groups are joined to form a heterocyclylgroup.

The term “alkoxycarbonylamino” refers to the group —NRC(O)OR where eachR is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl,or heterocyclyl wherein alkyl, substituted alkyl, aryl, heteroaryl, andheterocyclyl are as defined herein.

The term “acyloxy” refers to the groups alkyl-C(O)O—, substitutedalkyl-C(O)O—, cycloalkyl-C(O)O—, substituted cycloalkyl-C(O)O—,aryl-C(O)O—, heteroaryl-C(O)O—, and heterocyclyl-C(O)O— wherein alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl,and heterocyclyl are as defined herein.

“Aminosulfonyl” refers to the group —SO₂NR²¹R²², wherein R²¹ and R²²independently are selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic and where R²¹ and R²²are optionally joined together with the nitrogen bound thereto to form aheterocyclic or substituted heterocyclic group and alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Sulfonylamino” refers to the group —NR²¹SO₂R²², wherein R²¹ and R²²independently are selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R²¹ andR²² are optionally joined together with the atoms bound thereto to forma heterocyclic or substituted heterocyclic group, and wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are as definedherein.

“Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from6 to 18 carbon atoms having a single ring (such as is present in aphenyl group) or a ring system having multiple condensed rings (examplesof such aromatic ring systems include naphthyl, anthryl and indanyl)which condensed rings may or may not be aromatic, provided that thepoint of attachment is through an atom of an aromatic ring. This termincludes, by way of example, phenyl and naphthyl. Unless otherwiseconstrained by the definition for the aryl substituent, such aryl groupscan optionally be substituted with from 1 to 5 substituents, or from 1to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl,alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl,substituted alkoxy, substituted alkenyl, substituted alkynyl,substituted cycloalkyl, substituted cycloalkenyl, amino, substitutedamino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl,carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy,heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy,substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl and trihalomethyl.

“Aryloxy” refers to the group —O-aryl, wherein aryl is as definedherein, including, by way of example, phenoxy, naphthoxy, and the like,including optionally substituted aryl groups as also defined herein.

“Amino” refers to the group —NH₂.

The term “substituted amino” refers to the group —NRR where each R isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,substituted alkynyl, aryl, heteroaryl, and heterocyclyl provided that atleast one R is not hydrogen.

The term “azido” refers to the group —N₃.

“Carboxyl,” “carboxy” or “carboxylate” refers to —CO₂H or salts thereof.

“Carboxyl ester” or “carboxy ester” or the terms “carboxyalkyl” or“carboxylalkyl” refers to the groups —C(O)O-alkyl, —C(O)O-substitutedalkyl, —C(O)O-alkenyl, —C(O)O-substituted alkenyl, —C(O)O-alkynyl,—C(O)O-substituted alkynyl, —C(O)O-aryl, —C(O)O-substituted aryl,—C(O)O-cycloalkyl, —C(O)O-substituted cycloalkyl, —C(O)O-cycloalkenyl,—C(O)O-substituted cycloalkenyl, —C(O)O-heteroaryl, —C(O)O-substitutedheteroaryl, —C(O)O-heterocyclic, and —C(O)O-substituted heterocyclic,wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“(Carboxyl ester)oxy” or “carbonate” refers to the groups 0—C(O)O-alkyl,—O—C(O)O-substituted alkyl, —O—C(O)O-alkenyl, —O—C(O)O-substitutedalkenyl, —O—C(O)O-alkynyl, —O—C(O)O-substituted alkynyl, —O—C(O)O-aryl,—O—C(O)O-substituted aryl, —O—C(O)O-cycloalkyl, —O—C(O)O-substitutedcycloalkyl, —O—C(O)O-cycloalkenyl, —O—C(O)O-substituted cycloalkenyl,—O—C(O)O-heteroaryl, —O—C(O)O-substituted heteroaryl,—O—C(O)O-heterocyclic, and —O—C(O)O-substituted heterocyclic, whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“Cyano” or “nitrile” refers to the group —CN.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atomshaving single or multiple cyclic rings including fused, bridged, andspiro ring systems. Examples of suitable cycloalkyl groups include, forinstance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyland the like. Such cycloalkyl groups include, by way of example, singlering structures such as cyclopropyl, cyclobutyl, cyclopentyl,cyclooctyl, and the like, or multiple ring structures such asadamantanyl, and the like.

The term “substituted cycloalkyl” refers to cycloalkyl groups havingfrom 1 to 5 substituents, or from 1 to 3 substituents, selected fromalkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

“Cycloalkenyl” refers to non-aromatic cyclic alkyl groups of from 3 to10 carbon atoms having single or multiple rings and having at least onedouble bond and preferably from 1 to 2 double bonds.

The term “substituted cycloalkenyl” refers to cycloalkenyl groups havingfrom 1 to 5 substituents, or from 1 to 3 substituents, selected fromalkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano,halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy,thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

“Cycloalkynyl” refers to non-aromatic cycloalkyl groups of from 5 to 10carbon atoms having single or multiple rings and having at least onetriple bond.

“Cycloalkoxy” refers to —O-cycloalkyl.

“Cycloalkenyloxy” refers to —O-cycloalkenyl.

“Halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.

“Hydroxy” or “hydroxyl” refers to the group —OH.

“Heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms,such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected fromthe group consisting of oxygen, nitrogen, and sulfur within the ring.Such heteroaryl groups can have a single ring (such as, pyridinyl,imidazolyl or furyl) or multiple condensed rings in a ring system (forexample as in groups such as, indolizinyl, quinolinyl, benzofuran,benzimidazolyl or benzothienyl), wherein at least one ring within thering system is aromatic and at least one ring within the ring system isaromatic , provided that the point of attachment is through an atom ofan aromatic ring. In certain embodiments, the nitrogen and/or sulfurring atom(s) of the heteroaryl group are optionally oxidized to providefor the N-oxide (N→O), sulfinyl, or sulfonyl moieties. This termincludes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl,and furanyl. Unless otherwise constrained by the definition for theheteroaryl substituent, such heteroaryl groups can be optionallysubstituted with 1 to 5 substituents, or from 1 to 3 substituents,selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substitutedalkoxy, substituted alkenyl, substituted alkynyl, substitutedcycloalkyl, substituted cycloalkenyl, amino, substituted amino,aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl,carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy,heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy,substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl, andtrihalomethyl.

The term “heteroaralkyl” refers to the groups -alkylene-heteroaryl wherealkylene and heteroaryl are defined herein. This term includes, by wayof example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

“Heteroaryloxy” refers to —O-heteroaryl.

“Heterocycle,” “heterocyclic,” “heterocycloalkyl,” and “heterocyclyl”refer to a saturated or unsaturated group having a single ring ormultiple condensed rings, including fused bridged and spiro ringsystems, and having from 3 to 20 ring atoms, including 1 to 10 heteroatoms. These ring atoms are selected from the group consisting ofnitrogen, sulfur, or oxygen, wherein, in fused ring systems, one or moreof the rings can be cycloalkyl, aryl, or heteroaryl, provided that thepoint of attachment is through the non-aromatic ring. In certainembodiments, the nitrogen and/or sulfur atom(s) of the heterocyclicgroup are optionally oxidized to provide for the N-oxide, —S(O)—, or—SO₂— moieties.

Examples of heterocycles and heteroaryls include, but are not limitedto, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine,tetrahydrofuranyl, and the like.

Unless otherwise constrained by the definition for the heterocyclicsubstituent, such heterocyclic groups can be optionally substituted with1 to 5, or from 1 to 3 substituents, selected from alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl,—SO₂-heteroaryl, and fused heterocycle.

“Heterocyclyloxy” refers to the group —O-heterocyclyl.

The term “heterocyclylthio” refers to the group heterocyclic-S—.

The term “heterocyclene” refers to the diradical group formed from aheterocycle, as defined herein.

The term “hydroxyamino” refers to the group —NHOH.

“Nitro” refers to the group —NO₂.

“Oxo” refers to the atom (═O).

“Sulfonyl” refers to the group SO₂-alkyl, SO₂-substituted alkyl,SO₂-alkenyl, SO₂-substituted alkenyl, SO₂-cycloalkyl, SO₂-substitutedcylcoalkyl, SO₂-cycloalkenyl, SO₂-substituted cylcoalkenyl, SO₂-aryl,SO₂-substituted aryl, SO₂-heteroaryl, SO₂-substituted heteroaryl,SO₂-heterocyclic, and SO₂-substituted heterocyclic, wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are as definedherein. Sulfonyl includes, by way of example, methyl-SO₂—, phenyl-SO₂—,and 4-methylphenyl-SO₂—.

“Sulfonyloxy” refers to the group OSO₂-alkyl, OSO₂-substituted alkyl,OSO₂-alkenyl, OSO₂-substituted alkenyl, OSO₂-cycloalkyl,OSO₂-substituted cylcoalkyl, OSO₂-cycloalkenyl, OSO₂-substitutedcylcoalkenyl, OSO₂-aryl, OSO₂-substituted aryl, OSO₂-heteroaryl,OSO₂-substituted heteroaryl, OSO₂-heterocyclic, and OSO₂ substitutedheterocyclic, wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic are as defined herein.

The term “aminocarbonyloxy” refers to the group —OC(O)NRR where each Ris independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl,or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl andheterocyclic are as defined herein.

“Thiol” refers to the group —SH.

“Thioxo” or the term “thioketo” refers to the atom (═S).

“Alkylthio” or the term “thioalkoxy” refers to the group —S-alkyl,wherein alkyl is as defined herein. In certain embodiments, sulfur maybe oxidized to —S(O)—. The sulfoxide may exist as one or morestereoisomers.

The term “substituted thioalkoxy” refers to the group —S-substitutedalkyl.

The term “thioaryloxy” refers to the group aryl-S— wherein the arylgroup is as defined herein including optionally substituted aryl groupsalso defined herein.

The term “thioheteroaryloxy” refers to the group heteroaryl-S— whereinthe heteroaryl group is as defined herein including optionallysubstituted aryl groups as also defined herein.

The term “thioheterocyclooxy” refers to the group heterocyclyl-S—wherein the heterocyclyl group is as defined herein including optionallysubstituted heterocyclyl groups as also defined herein.

In addition to the disclosure herein, the term “substituted,” when usedto modify a specified group or radical, can also mean that one or morehydrogen atoms of the specified group or radical are each, independentlyof one another, replaced with the same or different substituent groupsas defined below.

In addition to the groups disclosed with respect to the individual termsherein, substituent groups for substituting for one or more hydrogens(any two hydrogens on a single carbon can be replaced with ═O, ═NR⁷⁰,═N—OR⁷⁰, ═N₂ or ═S) on saturated carbon atoms in the specified group orradical are, unless otherwise specified, —R⁶⁰, halo, ═O, —OR⁷⁰, —SR⁷⁰,—NR⁸⁰R⁸⁰, trihalomethyl, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —SO₂R⁷⁰,—SO₂O⁻M⁺, —SO₂OR⁷⁰, —OSO₂R⁷⁰, —OSO₂O⁻M⁺, —OSO₂OR⁷⁰, —P(O)(O⁻)₂(M^(±))₂,—P(O)(OR⁷⁰)O⁻M⁺, —P(O)(OR⁷⁰)₂, —C(O)R⁷⁰, —C(S)R⁷⁰, —C(NR⁷⁰)R⁷⁰,—C(O)O⁻M⁺, —C(O)OR⁷⁰, —C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰, —C(NR⁷⁰)NR⁸⁰R⁸⁰,—OC(O)R⁷⁰, —OC(S)R⁷⁰, —OC(O)O⁻M⁺, —OC(O)OR⁷⁰, —OC(S)OR⁷⁰, —NR⁷⁰C(O)R⁷⁰,—NR⁷⁰C(S)R⁷⁰, —NR⁷⁰CO₂ ⁻M⁺, —NR⁷⁰CO₂R⁷⁰, —NR⁷⁰C (S)OR⁷⁰,—NR⁷⁰C(O)NR⁸⁰R⁸⁰, —NR⁷⁰C(NR⁷⁰)R⁷⁰ and —NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰ isselected from the group consisting of optionally substituted alkyl,cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl,arylalkyl, heteroaryl and heteroarylalkyl, each R⁷⁰ is independentlyhydrogen or R⁶⁰; each R⁸⁰ is independently R⁷⁰ or alternatively, twoR^(80′)s, taken together with the nitrogen atom to which they arebonded, form a 5-, 6- or 7-membered heterocycloalkyl which mayoptionally include from 1 to 4 of the same or different additionalheteroatoms selected from the group consisting of O, N and S, of which Nmay have —H or C₁-C₃ alkyl substitution; and each M⁺ is a counter ionwith a net single positive charge. Each M⁺ may independently be, forexample, an alkali ion, such as K⁺, Na⁺, Li⁺; an ammonium ion, such as⁺N(R⁶⁰)₄; or an alkaline earth ion, such as [Ca²⁺]_(0.5), [Mg²⁺]_(0.5),or [Ba²⁺]_(0.5) (“subscript 0.5 means that one of the counter ions forsuch divalent alkali earth ions can be an ionized form of a compound ofthe invention and the other a typical counter ion such as chloride, ortwo ionized compounds disclosed herein can serve as counter ions forsuch divalent alkali earth ions, or a doubly ionized compound of theinvention can serve as the counter ion for such divalent alkali earthions). As specific examples, —NR⁸⁰R⁸⁰ is meant to include —NH₂,—NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl andN-morpholinyl.

In addition to the disclosure herein, substituent groups for hydrogenson unsaturated carbon atoms in “substituted” alkene, alkyne, aryl andheteroaryl groups are, unless otherwise specified, —R⁶⁰, halo, —O⁻M⁺,—OR⁷⁰, —SR⁷⁰, —NR⁸⁰R⁸⁰, trihalomethyl, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂,—N₃, —SO₂R⁷⁰, —SO₃ ⁻M⁺, —SO₃R⁷⁰, —OSO₂R⁷⁰, —OSO₃ ⁻M⁺, —OSO₃R⁷⁰, —PO₃⁻²(M⁺)₂, —P(O)(OR⁷⁰)O⁻M⁺, —P(O)(OR⁷⁰)₂, —C(O)R⁷⁰, —C(S)R⁷⁰, —C(NR⁷⁰)R⁷⁰,—CO₂ ⁻M⁺, —CO₂R⁷⁰, —C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰, —C(NR⁷⁰)NR⁸⁰R⁸⁰, —OC(O)R⁷⁰,—OC(S)R⁷⁰, —OCO₂ ⁻M⁺, —OCO₂R⁷⁰, —OC(S)OR⁷⁰, —NR⁷⁰C(O)R⁷⁰, —NR⁷⁰C(S)R⁷⁰,—NR⁷⁰CO₂ ⁻M⁺, —NR⁷⁰CO₂R⁷⁰, —NR⁷⁰C(S)OR⁷⁰, —NR⁷⁰C(O)NR⁸⁰R⁸⁰,—NR⁷⁰C(NR⁷⁰)R⁷⁰ and —NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰, R⁷⁰, R⁸⁰ and M⁺ areas previously defined, provided that in case of substituted alkene oralkyne, the substituents are not —O⁻M⁺, −OR⁷⁰, —SR⁷⁰, or —S⁻M⁺.

In addition to the groups disclosed with respect to the individual termsherein, substituent groups for hydrogens on nitrogen atoms in“substituted” heteroalkyl and cycloheteroalkyl groups are, unlessotherwise specified, —R⁶⁰, —O⁻M⁺, —OR⁷⁰, —SR⁷⁰, —NR⁸⁰R⁸⁰, trihalomethyl,—CF₃, —CN, —NO, —NO₂, —S(O)₂R⁷⁰, —S(O)₂O⁻M⁺, —S(O)₂OR⁷⁰, —OS(O)₂R⁷⁰,—OS(O)₂O⁻M⁺, —OS(O)₂OR⁷⁰, —P(O)(O)₂(M⁺)₂, —P(O)(OR⁷⁰)O⁻M⁺,—P(O)(OR⁷⁰)(OR⁷⁰), —C(O)R⁷⁰, —C(S)R⁷⁰, —C(NR⁷⁰)R⁷⁰, —C(O)OR⁷⁰,—C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰, -C(N12⁷⁰)NR⁸⁰R⁸⁰, —OC(O)R⁷⁰, —OC(S)R⁷⁰,—OC(O)OR⁷⁰, —OC(S)OR⁷⁰, —NR⁷⁰C(O)R⁷⁰, —NR⁷⁰C(S)R⁷⁰, —NR⁷⁰C(O)OR⁷⁰,—NR⁷⁰C(S)OR⁷⁰, —NR⁷⁰C(O)NR⁸⁰R⁸⁰, —NR⁷⁰C(NR⁷⁰)R⁷⁰ and—NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰, R⁷⁰, R⁸⁰ and M⁺ are as previouslydefined.

In addition to the disclosure herein, in a certain embodiment, a groupthat is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3substituents, 1 or 2 substituents, or 1 substituent.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,which is further substituted by a substituted aryl group, etc.) are notintended for inclusion herein. In such cases, the maximum number of suchsubstitutions is three. For example, serial substitutions of substitutedaryl groups specifically contemplated herein are limited to substitutedaryl-(substituted aryl)-substituted aryl.

Unless indicated otherwise, the nomenclature of substituents that arenot explicitly defined herein are arrived at by naming the terminalportion of the functionality followed by the adjacent functionalitytoward the point of attachment. For example, the substituent“arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O—C(O)—.

As to any of the groups disclosed herein which contain one or moresubstituents, it is understood, of course, that such groups do notcontain any substitution or substitution patterns which are stericallyimpractical and/or synthetically non-feasible. In addition, the subjectcompounds include all stereochemical isomers arising from thesubstitution of these compounds.

The term “pharmaceutically acceptable salt” means a salt which isacceptable for administration to a patient, such as a mammal (salts withcounterions having acceptable mammalian safety for a given dosageregime). Such salts can be derived from pharmaceutically acceptableinorganic or organic bases and from pharmaceutically acceptableinorganic or organic acids. “Pharmaceutically acceptable salt” refers topharmaceutically acceptable salts of a compound, which salts are derivedfrom a variety of organic and inorganic counter ions well known in theart and include, by way of example only, sodium, potassium, calcium,magnesium, ammonium, tetraalkylammonium, and the like; and when themolecule contains a basic functionality, salts of organic or inorganicacids, such as hydrochloride, hydrobromide, formate, tartrate, besylate,mesylate, acetate, maleate, oxalate, and the like.

The term “salt thereof” means a compound formed when a proton of an acidis replaced by a cation, such as a metal cation or an organic cation andthe like. Where applicable, the salt is a pharmaceutically acceptablesalt, although this is not required for salts of intermediate compoundsthat are not intended for administration to a patient. By way ofexample, salts of the present compounds include those wherein thecompound is protonated by an inorganic or organic acid to form a cation,with the conjugate base of the inorganic or organic acid as the anioniccomponent of the salt.

“Solvate” refers to a complex formed by combination of solvent moleculeswith molecules or ions of the solute. The solvent can be an organiccompound, an inorganic compound, or a mixture of both. Some examples ofsolvents include, but are not limited to, methanol,N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water.When the solvent is water, the solvate formed is a hydrate.

“Stereoisomer” and “stereoisomers” refer to compounds that have sameatomic connectivity but different atomic arrangement in space.Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers,and diastereomers.

“Tautomer” refers to alternate forms of a molecule that differ only inelectronic bonding of atoms and/or in the position of a proton, such asenol-keto and imine-enamine tautomers, or the tautomeric forms ofheteroaryl groups containing a —N═C(H)—NH— ring atom arrangement, suchas pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles. Aperson of ordinary skill in the art would recognize that othertautomeric ring atom arrangements are possible.

It will be appreciated that the term “or a salt or solvate orstereoisomer thereof” is intended to include all permutations of salts,solvates and stereoisomers, such as a solvate of a pharmaceuticallyacceptable salt of a stereoisomer of subject compound.

“Pharmaceutically effective amount” and “therapeutically effectiveamount” refer to an amount of a compound sufficient to treat a specifieddisorder or disease or one or more of its symptoms and/or to prevent theoccurrence of the disease or disorder. In reference to tumorigenicproliferative disorders, a pharmaceutically or therapeutically effectiveamount comprises an amount sufficient to, among other things, cause thetumor to shrink or decrease the growth rate of the tumor.

“Patient” refers to human and non-human subjects, especially mammaliansubjects.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition in a patient, such as amammal (particularly a human) that includes: (a) preventing the diseaseor medical condition from occurring, such as, prophylactic treatment ofa subject; (b) ameliorating the disease or medical condition, such as,eliminating or causing regression of the disease or medical condition ina patient; (c) suppressing the disease or medical condition, for exampleby, slowing or arresting the development of the disease or medicalcondition in a patient; or (d) alleviating a symptom of the disease ormedical condition in a patient.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymeric form of amino acids ofany length. Unless specifically indicated otherwise, “polypeptide,”“peptide,” and “protein” can include genetically coded and non-codedamino acids, chemically or biochemically modified or derivatized aminoacids, and polypeptides having modified peptide backbones. The termincludes fusion proteins, including, but not limited to, fusion proteinswith a heterologous amino acid sequence, fusions with heterologous andhomologous leader sequences, proteins which contain at least oneN-terminal methionine residue (e.g., to facilitate production in arecombinant bacterial host cell); immunologically tagged proteins; andthe like.

“Native amino acid sequence” or “parent amino acid sequence” are usedinterchangeably herein to refer to the amino acid sequence of apolypeptide prior to modification to include a modified amino acidresidue.

The terms “amino acid analog,” “unnatural amino acid,” and the like maybe used interchangeably, and include amino acid-like compounds that aresimilar in structure and/or overall shape to one or more amino acidscommonly found in naturally occurring proteins (e.g., Ala or A, Cys orC, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K,Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S,Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also includenatural amino acids with modified side chains or backbones. Amino acidanalogs also include amino acid analogs with the same stereochemistry asin the naturally occurring D-form, as well as the L-form of amino acidanalogs. In some instances, the amino acid analogs share backbonestructures, and/or the side chain structures of one or more naturalamino acids, with difference(s) being one or more modified groups in themolecule. Such modification may include, but is not limited to,substitution of an atom (such as N) for a related atom (such as S),addition of a group (such as methyl, or hydroxyl, etc.) or an atom (suchas Cl or Br, etc.), deletion of a group, substitution of a covalent bond(single bond for double bond, etc.), or combinations thereof. Forexample, amino acid analogs may include α-hydroxy acids, and α-aminoacids, and the like.

The term “carbohydrate” and the like may be used to refer to monomersunits and/or polymers of monosaccharides, disaccharides,oligosaccharides, and polysaccharides. The term sugar may be used torefer to the smaller carbohydrates, such as monosaccharides,disaccharides. The term “carbohydrate derivative” includes compoundswhere one or more functional groups of a carbohydrate of interest aresubstituted (replaced by any convenient substituent), modified(converted to another group using any convenient chemistry) or absent(e.g., eliminated or replaced by H). A variety of carbohydrates andcarbohydrate derivatives are available and may be adapted for use in thesubject compounds and conjugates.

The term “antibody” is used in the broadest sense and includesmonoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, and multispecific antibodies (e.g., bispecificantibodies), humanized antibodies, single-chain antibodies, chimericantibodies, antibody fragments (e.g., Fab fragments), and the like. Anantibody is capable of binding a target antigen. (Janeway, C., Travers,P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., GarlandPublishing, New York). A target antigen can have one or more bindingsites, also called epitopes, recognized by complementarity determiningregions (CDRs) formed by one or more variable regions of an antibody.

The term “natural antibody” refers to an antibody in which the heavy andlight chains of the antibody have been made and paired by the immunesystem of a multi-cellular organism. Spleen, lymph nodes, bone marrowand serum are examples of tissues that produce natural antibodies. Forexample, the antibodies produced by the antibody producing cellsisolated from a first animal immunized with an antigen are naturalantibodies.

The term “humanized antibody” or “humanized immunoglobulin” refers to anon-human (e.g., mouse or rabbit) antibody containing one or more aminoacids (in a framework region, a constant region or a CDR, for example)that have been substituted with a correspondingly positioned amino acidfrom a human antibody. In general, humanized antibodies produce areduced immune response in a human host, as compared to a non-humanizedversion of the same antibody. Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332). In certain embodiments, framework substitutions areidentified by modeling of the interactions of the CDR and frameworkresidues to identify framework residues important for antigen bindingand sequence comparison to identify unusual framework residues atparticular positions (see, e.g., U.S. Pat. No. 5,585,089; Riechmann etal., Nature 332:323 (1988)). Additional methods for humanizingantibodies contemplated for use in the present invention are describedin U.S. Pat. Nos. 5,750,078; 5,502,167; 5,705,154; 5,770,403; 5,698,417;5,693,493; 5,558,864; 4,935,496; and 4,816,567, and PCT publications WO98/45331 and WO 98/45332. In particular embodiments, a subject rabbitantibody may be humanized according to the methods set forth inUS20040086979 and US20050033031. Accordingly, the antibodies describedabove may be humanized using methods that are well known in the art.

The term “chimeric antibodies” refer to antibodies whose light and heavychain genes have been constructed, typically by genetic engineering,from antibody variable and constant region genes belonging to differentspecies. For example, the variable segments of the genes from a mousemonoclonal antibody may be joined to human constant segments, such asgamma 1 and gamma 3. An example of a therapeutic chimeric antibody is ahybrid protein composed of the variable or antigen-binding domain from amouse antibody and the constant or effector domain from a humanantibody, although domains from other mammalian species may be used.

By “genetically-encodable” as used in reference to an amino acidsequence of polypeptide, peptide or protein means that the amino acidsequence is composed of amino acid residues that are capable ofproduction by transcription and translation of a nucleic acid encodingthe amino acid sequence, where transcription and/or translation mayoccur in a cell or in a cell-free in vitro transcription/translationsystem.

The term “control sequences” refers to DNA sequences that facilitateexpression of an operably linked coding sequence in a particularexpression system, e.g. mammalian cell, bacterial cell, cell-freesynthesis, etc. The control sequences that are suitable for prokaryotesystems, for example, include a promoter, optionally an operatorsequence, and a ribosome binding site. Eukaryotic cell systems mayutilize promoters, polyadenylation signals, and enhancers.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate the initiation of translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading frame. Linking is accomplished by ligation or throughamplification reactions. Synthetic oligonucleotide adaptors or linkersmay be used for linking sequences in accordance with conventionalpractice.

The term “expression cassette” as used herein refers to a segment ofnucleic acid, usually DNA, that can be inserted into a nucleic acid(e.g., by use of restriction sites compatible with ligation into aconstruct of interest or by homologous recombination into a construct ofinterest or into a host cell genome). In general, the nucleic acidsegment comprises a polynucleotide that encodes a polypeptide ofinterest, and the cassette and restriction sites are designed tofacilitate insertion of the cassette in the proper reading frame fortranscription and translation. Expression cassettes can also compriseelements that facilitate expression of a polynucleotide encoding apolypeptide of interest in a host cell. These elements may include, butare not limited to: a promoter, a minimal promoter, an enhancer, aresponse element, a terminator sequence, a polyadenylation sequence, andthe like.

As used herein the term “isolated” is meant to describe a compound ofinterest that is in an environment different from that in which thecompound naturally occurs. “Isolated” is meant to include compounds thatare within samples that are substantially enriched for the compound ofinterest and/or in which the compound of interest is partially orsubstantially purified.

As used herein, the term “substantially purified” refers to a compoundthat is removed from its natural environment and is at least 60% free,at least 75% free, at least 80% free, at least 85% free, at least 90%free, at least 95% free, at least 98% free, or more than 98% free, fromother components with which it is naturally associated.

The term “physiological conditions” is meant to encompass thoseconditions compatible with living cells, e.g., predominantly aqueousconditions of a temperature, pH, salinity, etc. that are compatible withliving cells.

By “reactive partner” is meant a molecule or molecular moiety thatspecifically reacts with another reactive partner to produce a reactionproduct. Exemplary reactive partners include a cysteine or serine of asulfatase motif and Formylglycine Generating Enzyme (FGE), which reactto form a reaction product of a converted aldehyde tag containing aformylglycine (fGly) in lieu of cysteine or serine in the motif. Otherexemplary reactive partners include an aldehyde of an fGly residue of aconverted aldehyde tag (e.g., a reactive aldehyde group) and an“aldehyde-reactive reactive partner”, which comprises analdehyde-reactive group and a moiety of interest, and which reacts toform a reaction product of a modified aldehyde tagged polypeptide havingthe moiety of interest conjugated to the modified polypeptide through amodified fGly residue.

“N-terminus” refers to the terminal amino acid residue of a polypeptidehaving a free amine group, which amine group in non-N-terminus aminoacid residues normally forms part of the covalent backbone of thepolypeptide.

“C-terminus” refers to the terminal amino acid residue of a polypeptidehaving a free carboxyl group, which carboxyl group in non-C-terminusamino acid residues normally forms part of the covalent backbone of thepolypeptide.

By “internal site” as used in referenced to a polypeptide or an aminoacid sequence of a polypeptide means a region of the polypeptide that isnot at the N-terminus or at the C-terminus.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed, to the extent that suchcombinations embrace subject matter that are, for example, compoundsthat are stable compounds (i.e., compounds that can be made, isolated,characterized, and tested for biological activity). In addition, allsub-combinations of the various embodiments and elements thereof (e.g.,elements of the chemical groups listed in the embodiments describingsuch variables) are also specifically embraced by the present inventionand are disclosed herein just as if each and every such sub-combinationwas individually and explicitly disclosed herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides conjugates (e.g., polypeptideconjugates), hydrazinyl-pyrrolo compounds for producing the conjugatesand methods of making and using the same. Embodiments of each aredescribed in more detail in the sections below.

Conjugates

The present disclosure provides conjugates. By “conjugate” is meant afirst moiety that is stably associated with a second moiety. By “stablyassociated” is meant that a moiety is bound to another moiety orstructure under standard conditions. In certain embodiments, the firstand second moieties are bound to each other through one or more covalentbonds.

In certain embodiments, the conjugate is a polypeptide conjugate, whichincludes a polypeptide conjugated to a second moiety. As described inmore detail below, the moiety conjugated to the polypeptide can be anyof a variety of moieties such as, but not limited to, a detectablelabel, a drug, a water-soluble polymer, or a moiety for immobilizationof the polypeptide to a membrane or a surface. The moiety of interestcan be conjugated to the polypeptide at any desired site of thepolypeptide. Thus, the present disclosure provides, for example, amodified polypeptide having a moiety conjugated at a site at or near theC-terminus of the polypeptide. Other examples include a modifiedpolypeptide having a moiety conjugated at a position at or near theN-terminus of the polypeptide. Examples also include a modifiedpolypeptide having a moiety conjugated at a position between theC-terminus and the N-terminus of the polypeptide (e.g., at an internalsite of the polypeptide). Combinations of the above are also possiblewhere the modified polypeptide is conjugated to two or more moieties.

Embodiments of the present disclosure include conjugates where apolypeptide is conjugated to one or more moieties, such as 2 moieties, 3moieties, 4 moieties, 5 moieties, 6 moieties, 7 moieties, 8 moieties, 9moieties, or 10 or more moieties. The moieties may be conjugated to thepolypeptide at one or more sites in the polypeptide. For example, one ormore moieties may be conjugated to a single amino acid residue of thepolypeptide. In some cases, one moiety is conjugated to an amino acidresidue of the polypeptide. In other embodiments, two moieties may beconjugated to the same amino acid residue of the polypeptide. In otherembodiments, a first moiety is conjugated to a first amino acid residueof the polypeptide and a second moiety is conjugated to a second aminoacid residue of the polypeptide. Combinations of the above are alsopossible, for example where a polypeptide is conjugated to a firstmoiety at a first amino acid residue and conjugated to two othermoieties at a second amino acid residue. Other combinations are alsopossible, such as, but not limited to, a polypeptide conjugated to firstand second moieties at a first amino acid residue and conjugated tothird and fourth moieties at a second amino acid residue, etc.

The one or more amino acid residues that are conjugated to the one ormore moieties may be naturally occurring amino acids, unnatural aminoacids, or combinations thereof. For instance, the conjugate may includea moiety conjugated to a naturally occurring amino acid residue of thepolypeptide. In other instances, the conjugate may include a moietyconjugated to an unnatural amino acid residue of the polypeptide. One ormore moieties may be conjugated to the polypeptide at a single naturalor unnatural amino acid residue as described above. One or more naturalor unnatural amino acid residues in the polypeptide may be conjugated tothe moiety or moieties as described herein. For example, two (or more)amino acid residues (e.g., natural or unnatural amino acid residues) inthe polypeptide may each be conjugated to one or two moieties, such thatmultiple sites in the polypeptide are modified.

Although described herein in terms of a polypeptide conjugated to one ormore moieties (e.g., a chemical entity, a polypeptide, etc.),embodiments of the present disclosure also include conjugates where amoiety (e.g., a chemical entity, such as a drug or a detectable label)is conjugated to one or more other moieties (e.g., a chemical entity, apolypeptide, etc.). For example, a drug may be conjugated to one or moreother moieties (e.g., a chemical entity, a polypeptide, etc.), or inother embodiments, a detectable label may be conjugated to one or moreother moieties (e.g., a chemical entity, a polypeptide, etc.). Thus, forinstance, embodiments of the present disclosure include, but are notlimited to, the following: a conjugate of a polypeptide and a drug; aconjugate of a polypeptide and a detectable label; a conjugate of two ormore polypeptides; a conjugate of two or more drugs; a conjugate of twoof more detectable labels; a conjugate of a drug and a detectable label;a conjugate of a polypeptide, a drug and a detectable label; a conjugateof a polypeptide and two or more drugs; a conjugate of a polypeptide andtwo or more detectable labels; a conjugate of a drug and two or morepolypeptides; a conjugate of a detectable label and two or morepolypeptides; and the like.

In certain embodiments, the polypeptide and the moiety of interest areconjugated through a coupling moiety. For example, the polypeptide andthe moiety of interest may each be bound (e.g., covalently bonded) tothe coupling moiety, thus indirectly binding the polypeptide and themoiety of interest together through the coupling moiety. In some cases,the coupling moiety includes a hydrazinyl-pyrrolo compound or aderivative of a hydrazinyl-pyrrolo compound. For instance, a generalscheme for coupling a moiety of interest to a polypeptide through ahydrazinyl-pyrrolo coupling moiety is shown in the general reactionscheme below.

In the reaction scheme above, R may be the moiety of interest conjugatedto the polypeptide. As described herein, the moiety can be any of avariety of moieties such as, but not limited to, chemical entity, suchas a detectable label, a drug, a water-soluble polymer, or a moiety forimmobilization of the polypeptide to a membrane or a surface of asubstrate. R′ and R″ may each independently be any desired substituent,such as, but not limited to, hydrogen, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Z¹,Z², Z³ and Z⁴ may be CR¹¹, NR¹², N, O or S, wherein one of Z¹, Z², Z³and Z⁴ is optional and R¹¹ and R¹² may be any desired substituent.

Other hydrazinyl-pyrrolo coupling moieties are also possible. Forexample, another general scheme for coupling a moiety of interest to apolypeptide through a hydrazinyl-pyrrolo coupling moiety is shown in thegeneral reaction scheme below.

In the reaction scheme above, R may be the moiety of interest conjugatedto the polypeptide. As described above, the moiety can be any of avariety of moieties such as, but not limited to, a chemical entity, suchas a detectable label, a drug, a water-soluble polymer, or a moiety forimmobilization of the polypeptide to a membrane or a surface of asubstrate. R′ and R″ may each independently be any desired substituent,such as, but not limited to, hydrogen, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Z¹,Z², Z³ and Z⁴ may be CR¹¹, NR¹², N, O, C or S, wherein one of Z¹, Z², Z³and Z⁴ is optional and R¹¹ and R¹² may be any desired substituent. Othercoupling moieties are also possible, as shown in the conjugates andcompounds described in more detail below.

In certain embodiments, the conjugate includes at least one modifiedamino acid residue of the formula (I):

wherein

m is 0 or 1;

R¹ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, and substituted heterocyclyl;

R² and R³ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, orR² and R³ are optionally cyclically linked to form a 5 or 6-memberedheterocyclyl;

Z¹, Z², Z³ and Z⁴ are each independently selected from CR¹¹, NR¹², O, Nand S, wherein one of Z¹, Z², Z³ and Z⁴ is optional;

X⁵ is C;

Y⁵, R¹¹ and R¹² are each independently selected from hydrogen, halogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl,alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substitutedthioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl;

Q¹ is a bond to either Z⁴ or X⁵, wherein if Q¹ is a bond to Z⁴, then Z⁴is CR¹¹ or NR¹² and R¹¹ or R¹² is absent, or if Q¹ is a bond to X⁵, thenY⁵ is absent;

R¹⁵ is -L-W¹ or -L-W¹ is attached to one of Z¹, Z², Z³ or Z⁴, wherein if-L-W¹ is attached to one of Z¹, Z², Z³ or Z⁴, then R¹⁵ is selected fromhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl;

L is a linker (e.g., a linker as described herein), and

one of W¹ and W² is a polypeptide and the other is a chemical entity.

In certain embodiments, m is 0 or 1. In certain embodiments, m is 0. Incertain embodiments, m is 1.

In certain embodiments, R¹ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R¹ is hydrogen. In certain embodiments, R¹ is alkylor substituted alkyl. In certain embodiments, R¹ is alkenyl orsubstituted alkenyl. In certain embodiments, R¹ is alkynyl orsubstituted alkynyl. In certain embodiments, R¹ is aryl or substitutedaryl. In certain embodiments, R¹ is heteroaryl or substitutedheteroaryl. In certain embodiments, R¹ is cycloalkyl or substitutedcycloalkyl. In certain embodiments, R¹ is heterocyclyl or substitutedheterocyclyl.

In certain embodiments, R² is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R² is hydrogen. In certain embodiments, R² is alkylor substituted alkyl. In certain embodiments, R² is alkenyl orsubstituted alkenyl. In certain embodiments, R² is alkynyl orsubstituted alkynyl. In certain embodiments, R² is alkoxy or substitutedalkoxy. In certain embodiments, R² is amino or substituted amino. Incertain embodiments, R² is carboxyl or carboxyl ester. In certainembodiments, R² is acyl or acyloxy. In certain embodiments, R² is acylamino or amino acyl. In certain embodiments, R² is alkylamide orsubstituted alkylamide. In certain embodiments, R² is sulfonyl. Incertain embodiments, R² is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R² is aryl or substituted aryl. In certainembodiments, R² is heteroaryl or substituted heteroaryl. In certainembodiments, R² is cycloalkyl or substituted cycloalkyl. In certainembodiments, R² is heterocyclyl or substituted heterocyclyl.

In certain embodiments, R² is alkyl or substituted alkyl. For example,R² may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R² is methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R² ismethyl.

In certain embodiments, R³ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R³ is hydrogen. In certain embodiments, R³ is alkylor substituted alkyl. In certain embodiments, R³ is alkenyl orsubstituted alkenyl. In certain embodiments, R³ is alkynyl orsubstituted alkynyl. In certain embodiments, R³ is alkoxy or substitutedalkoxy. In certain embodiments, R³ is amino or substituted amino. Incertain embodiments, R³ is carboxyl or carboxyl ester. In certainembodiments, R³ is acyl or acyloxy. In certain embodiments, R³ is acylamino or amino acyl. In certain embodiments, R³ is alkylamide orsubstituted alkylamide. In certain embodiments, R³ is sulfonyl. Incertain embodiments, R³ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R³ is aryl or substituted aryl. In certainembodiments, R³ is heteroaryl or substituted heteroaryl. In certainembodiments, R³ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R³ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, R³ is alkyl or substituted alkyl. For example,R³ may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R³ is methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R³ ismethyl.

In certain embodiments, R² and R³ are each independently selected fromalkyl and substituted alkyl. For example, R² may be alkyl or substitutedalkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆alkyl or C₁-C₆ substituted alkyl), and R³ may be alkyl or substitutedalkyl, such as, C₁-C_(1o) alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆alkyl or C₁-C₆ substituted alkyl). In some cases, R² and R³ are eachindependently selected from methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R²and R³ are each methyl.

In certain embodiments, R² and R³ are optionally cyclically linked toform a 5 or 6-membered heterocyclyl. In some instances, R² and R³(together with the atoms to which they are attached) may be cyclicallylinked to form a 5-membered heterocyclyl. In some instances, R² and R³(together with the atoms to which they are attached) may be cyclicallylinked to form a 6-membered heterocyclyl. For example, R² and R³ mayeach independently be an alkyl or substituted alkyl, such as, C₁-C₁₀alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆substituted alkyl), where R² and R³ are optionally cyclically linked toform a 5 or 6-membered heterocyclyl, as described above. In someinstances, one or more carbon atoms in R² and/or R³ may be replaced witha heteroatom, such as N, O, or S.

In certain embodiments, Z¹ is selected from CR¹¹, NR¹², O, N and S. Incertain embodiments, Z¹ is CR¹¹. In certain embodiments, Z¹ is NR¹². Incertain embodiments, Z¹ is N. In certain embodiments, Z¹ is O. Incertain embodiments, Z¹ is S.

In certain embodiments, Z² is selected from CR¹¹, NR¹², O, N and S. Incertain embodiments, Z² is CR¹¹. In certain embodiments, Z² is NR¹². Incertain embodiments, Z² is N. In certain embodiments, Z² is O. Incertain embodiments, Z² is S.

In certain embodiments, Z³ is selected from CR¹¹, NR¹², O, N and S. Incertain embodiments, Z³ is CR¹¹. In certain embodiments, Z³ is NR¹². Incertain embodiments, Z³ is N.

In certain embodiments, Z³ is O. In certain embodiments, Z³ is S.

In certain embodiments, Z⁴ is selected from CR¹¹, NR¹², O, N and S. Incertain embodiments, Z⁴ is CR¹¹. In certain embodiments, Z⁴ is NR¹². Incertain embodiments, Z⁴ is N. In certain embodiments, Z⁴ is O. Incertain embodiments, Z⁴ is S.

Various combinations of Z¹, Z², Z³ and Z⁴ are possible. For example, incertain embodiments, each of Z¹, Z², Z³ and Z⁴ is CR¹¹. In otherinstances, three of Z¹, Z², Z³ and Z⁴ are CR¹¹ and one of Z¹, Z², Z³ andZ⁴ is N. In other embodiments, two of Z¹, Z², Z³ and Z⁴ are CR¹¹ and twoof Z¹, Z², Z³ and Z⁴ are N. In other embodiments, one of Z¹, Z², Z³ andZ⁴ is CR¹¹ and three of Z¹, Z², Z³ and Z⁴ are N. In other embodiments,one of Z¹, Z², Z³ and Z⁴ is absent, two of Z¹, Z², Z³ and Z⁴ are CR¹¹and one of Z¹, Z², Z³ and Z⁴ is NR¹². In other embodiments, one of Z¹,Z², Z³ and Z⁴ is absent, two of Z¹, Z², Z³ and Z⁴ are CR¹¹ and one ofZ¹, Z², Z³ and Z⁴ is S. In other embodiments, one of Z¹, Z², Z³ and Z⁴is absent, two of Z¹, Z², Z³ and Z⁴ are CR¹¹ and one of Z¹, Z², Z³ andZ⁴ is O. Other combinations of CR¹¹, N, O and S are possible for Z¹, Z²,Z³ and Z⁴ as desired.

In certain embodiments, X⁵ is C.

In certain embodiments, each R¹¹ (if present) is independently selectedfrom hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, amino acyl, alkylamide, substituted alkylamide, sulfonyl,thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl. In certain embodiments, R¹¹is hydrogen. In certain embodiments, R¹¹ is halogen, such as F, Cl, Bror I. In certain embodiments, R¹¹ is F. In certain embodiments, R¹¹ isCl. In certain embodiments, R¹¹ is Br. In certain embodiments, R¹¹ is I.In certain embodiments, R¹¹ is alkyl or substituted alkyl. In certainembodiments, R¹¹ is alkenyl or substituted alkenyl. In certainembodiments, R¹¹ is alkynyl or substituted alkynyl. In certainembodiments, R¹¹ is alkoxy or substituted alkoxy. In certainembodiments, R¹¹ is amino or substituted amino. In certain embodiments,R¹¹ is carboxyl or carboxyl ester. In certain embodiments, R¹¹ is acylor acyloxy. In certain embodiments, R¹¹ is acyl amino or amino acyl. Incertain embodiments, R¹¹ is alkylamide or substituted alkylamide. Incertain embodiments, R¹¹ is sulfonyl. In certain embodiments, R¹¹ isthioalkoxy or substituted thioalkoxy. In certain embodiments, R¹¹ isaryl or substituted aryl. In certain embodiments, R¹¹ is heteroaryl orsubstituted heteroaryl. In certain embodiments, R¹¹ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, R¹¹ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, each R¹² (if present) is independently selectedfrom hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, amino acyl, alkylamide, substituted alkylamide, sulfonyl,thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl. In certain embodiments, R¹²is hydrogen. In certain embodiments, R¹² is halogen, such as F, Cl, Bror I. In certain embodiments, R¹² is F. In certain embodiments, R¹² isCl. In certain embodiments, R¹² is Br. In certain embodiments, R¹² is I.In certain embodiments, R¹² is alkyl or substituted alkyl. In certainembodiments, R¹² is alkenyl or substituted alkenyl. In certainembodiments, R¹² is alkynyl or substituted alkynyl. In certainembodiments, R¹² is alkoxy or substituted alkoxy. In certainembodiments, R¹² is amino or substituted amino. In certain embodiments,R¹² is carboxyl or carboxyl ester. In certain embodiments, R¹² is acylor acyloxy. In certain embodiments, R¹² is acyl amino or amino acyl. Incertain embodiments, R¹² is alkylamide or substituted alkylamide. Incertain embodiments, R¹² is sulfonyl. In certain embodiments, R¹² isthioalkoxy or substituted thioalkoxy. In certain embodiments, R¹² isaryl or substituted aryl. In certain embodiments, R¹² is heteroaryl orsubstituted heteroaryl. In certain embodiments, R¹² is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, R¹² is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y⁵ (if present) is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y⁵ is hydrogen. Incertain embodiments, Y⁵ is halogen, such as F, Cl, Br or I. In certainembodiments, Y⁵ is F. In certain embodiments, Y⁵ is Cl. In certainembodiments, Y⁵ is Br. In certain embodiments, Y⁵ is I. In certainembodiments, Y⁵ is alkyl or substituted alkyl. In certain embodiments,Y⁵ is alkenyl or substituted alkenyl. In certain embodiments, Y⁵ isalkynyl or substituted alkynyl. In certain embodiments, Y⁵ is alkoxy orsubstituted alkoxy. In certain embodiments, Y⁵ is amino or substitutedamino. In certain embodiments, Y⁵ is carboxyl or carboxyl ester. Incertain embodiments, Y⁵ is acyl or acyloxy. In certain embodiments, Y⁵is acyl amino or amino acyl. In certain embodiments, Y⁵ is alkylamide orsubstituted alkylamide. In certain embodiments, Y⁵ is sulfonyl. Incertain embodiments, Y⁵ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y⁵ is aryl or substituted aryl. In certainembodiments, Y⁵ is heteroaryl or substituted heteroaryl. In certainembodiments, Y⁵ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y⁵ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, Q¹ is a bond to either Z⁴ or X⁵. In certainembodiments, Q¹ is a bond to Z⁴. In certain embodiments, if Q¹ is a bondto Z⁴, then Z⁴ is CR¹¹ and R¹¹ is absent. In certain embodiments, if Q¹is a bond to Z⁴, then Z⁴ is NR¹² and R¹² is absent. In certainembodiments, Q¹ is a bond to X⁵. In certain embodiments, if Q¹ is a bondto X⁵, then Y⁵ is absent.

In certain embodiments, R¹⁵ is -L-W¹ or -L-W¹ is attached to one of Z¹,Z², Z³ or Z⁴. In certain embodiments, R¹⁵ is -L-W¹. In certainembodiments, R¹⁵ is not -L-W¹. In certain embodiments, -L-W¹ is attachedto one of Z¹, Z², Z³ or Z⁴. In certain embodiments, -L-W¹ is attached toZ¹. In certain embodiments, -L-W¹ is attached to Z². In certainembodiments, -L-W¹ is attached to Z³. In certain embodiments, -L-W¹ isattached to Z⁴.

In certain embodiments, if -L-W¹ is attached to one of Z¹, Z², Z³ or Z⁴,then R¹⁵ is is not -L-W¹. In these embodiments, R¹⁵ is selected fromhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl. In some instances, R¹⁵ ishydrogen. In some instances, R¹⁵ is alkyl or substituted alkyl. In someinstances, R¹⁵ is alkenyl or substituted alkenyl. In some instances, R¹⁵is alkynyl or substituted alkynyl. In some instances, R¹⁵ is aryl orsubstituted aryl. In some instances, R¹⁵ is heteroaryl or substitutedheteroaryl. In some instances, R¹⁵ is cycloalkyl or substitutedcycloalkyl. In some instances, R¹⁵ is heterocyclyl or substitutedheterocyclyl.

In certain embodiments, L is an optional linker. In certain embodiments,L is not present, and thus the nitrogen of the indole ring is directlybonded to W¹. In certain embodiments, L is present, and thus thenitrogen of the indole ring is indirectly bonded to W¹ through thelinker L. Further description of the linker, L, is found in thedisclosure herein.

For instance, in certain embodiments, L includes a group selected fromalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl amino, alkylamide, substitutedalkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl. In certain embodiments, L includes an alkyl or substitutedalkyl group. In certain embodiments, L includes an alkenyl orsubstituted alkenyl group. In certain embodiments, L includes an alkynylor substituted alkynyl group. In certain embodiments, L includes analkoxy or substituted alkoxy group. In certain embodiments, L includesan amino or substituted amino group. In certain embodiments, L includesa carboxyl or carboxyl ester group. In certain embodiments, L includesan acyl amino group. In certain embodiments, L includes an alkylamide orsubstituted alkylamide group. In certain embodiments, L includes an arylor substituted aryl group. In certain embodiments, L includes aheteroaryl or substituted heteroaryl group. In certain embodiments, Lincludes a cycloalkyl or substituted cycloalkyl group. In certainembodiments, L includes a heterocyclyl or substituted heterocyclylgroup.

In certain embodiments, L includes a polymer. For example, the polymermay include a polyalkylene glycol and derivatives thereof, includingpolyethylene glycol, methoxypolyethylene glycol, polyethylene glycolhomopolymers, polypropylene glycol homopolymers, copolymers of ethyleneglycol with propylene glycol (e.g., where the homopolymers andcopolymers are unsubstituted or substituted at one end with an alkylgroup), polyvinyl alcohol, polyvinyl ethyl ethers, polyvinylpyrrolidone,combinations thereof, and the like. In certain embodiments, the polymeris a polyalkylene glycol. In certain embodiments, the polymer is apolyethylene glycol.

In some embodiments, L is a linker comprising-(L1)_(a)-(L²)_(b)-(L³)_(c)-(L⁴)_(d)-(L⁵)_(e)-, wherein L¹, L² , L³, L⁴and L⁵ are each a linker unit, and a, b, c, d and e are eachindependently 0 or 1, wherein the sum of a, b, c, d and e is 1 to 5.Other linkers are also possible, as shown in the conjugates andcompounds described in more detail below.

In certain embodiments, W¹ is selected from a polypeptide and a chemicalentity. In certain embodiments, W¹ is a chemical entity. In certainembodiments, the chemical entity is a drug. In certain embodiments, thechemical entity is a detectable label. In certain embodiments, W¹ isselected from a detectable label and a polypeptide. In certainembodiments, W¹ is selected from a drug, a detectable label and apolypeptide. In certain embodiments, W¹ is a polypeptide.

In certain embodiments, W² is selected from a drug and a chemicalentity. In certain embodiments, W² is a chemical entity. In certainembodiments, the chemical entity is a drug. In certain embodiments, thechemical entity is a detectable label. In certain embodiments, W² isselected from a drug, a detectable label and a polypeptide. In certainembodiments, W² is a drug. In certain embodiments, W² is a detectablelabel. In certain embodiments, W² is a polypeptide.

In certain embodiments, one of W¹ and W² is a polypeptide and the otheris a chemical entity. In certain embodiments, the chemical entity is adrug. In certain embodiments, the chemical entity is a detectable label.In certain embodiments, W¹ is the chemical entity, and W² is thepolypeptide. In certain embodiments, W¹ is the polypeptide, and W² isthe chemical entity.

In certain embodiments, the conjugate includes at least one modifiedamino acid residue of the formula (II):

wherein

m, R¹, R², R³′ X⁵, L, Q¹, W¹, W², Y⁵, Z¹, Z², Z³ and Z⁴ are as definedin formula (I).

As described above, in formula (I), in some instances, R¹⁵ is -L-W¹,which results in a modified amino acid residue of formula (II) as shownabove.

In certain embodiments, the substituents for formula (II) are the sameas for formula (I) described above. For example, in certain embodiments,m is 0 or 1. In certain embodiments, m is 0. In certain embodiments, mis 1. In certain embodiments, the substituents for formula (II), e.g.,m, R¹, R², R³′ X⁵, L, Q¹, W¹, W², Y⁵, Z¹, Z², Z³ and Z⁴ are as definedin formula (I).

In some embodiments, the conjugate includes at least one modified aminoacid residue of formula (III):

wherein

m, R¹, R², R³′ X⁵, Y⁵, L, W¹ and W² are as defined in formula (I);

X¹, X², X³ and X⁴ are each independently selected from C, N, O and S,wherein one of X¹, X², X³ and X⁴ is optional;

Y¹, Y², Y³, Y⁴ and Y⁵, if present, are each independently selected fromhydrogen, halogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, amino acyl, alkylamide, substituted alkylamide, sulfonyl,thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, wherein Y¹ and Y², Y² andY³, or Y³ and Y⁴ are optionally cyclically linked; and

Q¹ is a bond to either X⁴ or X⁵, wherein if Q¹ is a bond to X⁴, then Y⁴is absent, or if Q¹ is a bond to X⁵, then Y⁵ is absent.

In certain embodiments, the substituents for formula (III) are the sameas for formula (I) described above. For example, in certain embodiments,m is 0 or 1. In certain embodiments, m is 0. In certain embodiments, mis 1. In certain embodiments, the substituents for formula (III), e.g.,m, R¹, R², R³′ X⁵, Y⁵, L, W¹ and W², are as defined in formula (I).

In certain embodiments, X¹ is selected from C, N, O and S. In certainembodiments, X¹ is C. In certain embodiments, X¹ is N. In certainembodiments, X¹ is O. In certain embodiments, X¹ is S.

In certain embodiments, X² is selected from C, N, O and S. In certainembodiments, X² is C. In certain embodiments, X² is N. In certainembodiments, X² is O. In certain embodiments, X² is S.

In certain embodiments, X³ is selected from C, N, O and S. In certainembodiments, X³ is C. In certain embodiments, X³ is N. In certainembodiments, X³ is O. In certain embodiments, X³ is S.

In certain embodiments, X⁴ is selected from C, N, O and S. In certainembodiments, X⁴ is C. In certain embodiments, X⁴ is N. In certainembodiments, X⁴ is O. In certain embodiments, X⁴ is S.

In certain embodiments, one of X¹, X², X³ and X⁴ is optional. In certainembodiments, X¹ is absent. In certain embodiments, X² is absent. Incertain embodiments, X³ is absent. In certain embodiments, X⁴ is absent.

Various combinations of X¹, X², X³ and X⁴ are possible. For example, incertain embodiments, each of X¹, X², X³ and X⁴ is C. In other instances,three of X¹, X², X³ and X⁴ are C and one of X¹, X², X³ and X⁴ is N. Inother embodiments, two of X¹, X², X³ and X⁴ are C and two of X¹, X², X³and X⁴ are N. In other embodiments, one of X¹, X², X³ and X⁴ is C andthree of X¹, X², X³ and X⁴ is are N. Other combinations of C, N, O and Sare possible for X¹, X², X³ and X⁴ as desired.

In certain embodiments, Y¹ (if present) is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y¹ is hydrogen. Incertain embodiments, Y¹ is halogen, such as F, Cl, Br or I. In certainembodiments, Y¹ is F. In certain embodiments, Y¹ is Cl. In certainembodiments, Y¹ is Br. In certain embodiments, Y¹ is I. In certainembodiments, Y¹ is alkyl or substituted alkyl. In certain embodiments,Y¹ is alkenyl or substituted alkenyl. In certain embodiments, Y¹ isalkynyl or substituted alkynyl. In certain embodiments, Y¹ is alkoxy orsubstituted alkoxy. In certain embodiments, Y¹ is amino or substitutedamino. In certain embodiments, Y¹ is carboxyl or carboxyl ester. Incertain embodiments, Y¹ is acyl or acyloxy. In certain embodiments, Y¹is acyl amino or amino acyl. In certain embodiments, Y¹ is alkylamide orsubstituted alkylamide. In certain embodiments, Y¹ is sulfonyl. Incertain embodiments, Y¹ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y¹ is aryl or substituted aryl. In certainembodiments, Y¹ is heteroaryl or substituted heteroaryl. In certainembodiments, Y¹ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y¹ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, Y² (if present) is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y² is hydrogen. Incertain embodiments, Y² is halogen, such as F, Cl, Br or I. In certainembodiments, Y² is F. In certain embodiments, Y² is Cl. In certainembodiments, Y² is Br. In certain embodiments, Y² is I. In certainembodiments, Y² is alkyl or substituted alkyl. In certain embodiments,Y² is alkenyl or substituted alkenyl. In certain embodiments, Y² isalkynyl or substituted alkynyl. In certain embodiments, Y² is alkoxy orsubstituted alkoxy. In certain embodiments, Y² is amino or substitutedamino. In certain embodiments, Y² is carboxyl or carboxyl ester. Incertain embodiments, Y² is acyl or acyloxy. In certain embodiments, Y²is acyl amino or amino acyl. In certain embodiments, Y² is alkylamide orsubstituted alkylamide. In certain embodiments, Y² is sulfonyl. Incertain embodiments, Y² is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y² is aryl or substituted aryl. In certainembodiments, Y² is heteroaryl or substituted heteroaryl. In certainembodiments, Y² is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y² is heterocyclyl or substituted heterocyclyl.

In certain embodiments, Y³ (if present) is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y³ is hydrogen. Incertain embodiments, Y³ is halogen, such as F, Cl, Br or I. In certainembodiments, Y³ is F. In certain embodiments, Y³ is Cl. In certainembodiments, Y³ is Br. In certain embodiments, Y³ is I. In certainembodiments, Y³ is alkyl or substituted alkyl. In certain embodiments,Y³ is alkenyl or substituted alkenyl. In certain embodiments, Y³ isalkynyl or substituted alkynyl. In certain embodiments, Y³ is alkoxy orsubstituted alkoxy. In certain embodiments, Y³ is amino or substitutedamino. In certain embodiments, Y³ is carboxyl or carboxyl ester. Incertain embodiments, Y³ is acyl or acyloxy. In certain embodiments, Y³is acyl amino or amino acyl. In certain embodiments, Y³ is alkylamide orsubstituted alkylamide. In certain embodiments, Y³ is sulfonyl. Incertain embodiments, Y³ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y³ is aryl or substituted aryl. In certainembodiments, Y³ is heteroaryl or substituted heteroaryl. In certainembodiments, Y³ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y³ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, Y⁴ (if present) is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y⁴ is hydrogen. Incertain embodiments, Y⁴ is halogen, such as F, Cl, Br or I. In certainembodiments, Y⁴ is F. In certain embodiments, Y⁴ is Cl. In certainembodiments, Y⁴ is Br. In certain embodiments, Y⁴ is I. In certainembodiments, Y⁴ is alkyl or substituted alkyl. In certain embodiments,Y⁴ is alkenyl or substituted alkenyl. In certain embodiments, Y⁴ isalkynyl or substituted alkynyl. In certain embodiments, Y⁴ is alkoxy orsubstituted alkoxy. In certain embodiments, Y⁴ is amino or substitutedamino. In certain embodiments, Y⁴ is carboxyl or carboxyl ester. Incertain embodiments, Y⁴ is acyl or acyloxy. In certain embodiments, Y⁴is acyl amino or amino acyl. In certain embodiments, Y⁴ is alkylamide orsubstituted alkylamide. In certain embodiments, Y⁴ is sulfonyl. Incertain embodiments, Y⁴ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y⁴ is aryl or substituted aryl. In certainembodiments, Y⁴ is heteroaryl or substituted heteroaryl. In certainembodiments, Y⁴ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y⁴ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, Y¹ and Y², Y² and Y³, or Y³ and Y⁴ arecyclically linked to form a fused benzo ring. In certain embodiments, Y¹and Y² are cyclically linked to form a fused benzo ring. In certainembodiments of formula, Y² and Y³ are cyclically linked to form a fusedbenzo ring. In certain embodiments of formula, Y³ and Y⁴ are cyclicallylinked to form a fused benzo ring.

In certain embodiments, Q¹ is a bond to either X⁴ or X⁵, wherein if Q¹is a bond to X⁴, then Y⁴ is absent, or if Q¹ is a bond to X⁵, then Y⁵ isabsent. In certain embodiments, Q¹ is a bond to X⁴, wherein if Q¹ is abond to X⁴, then Y⁴ is absent. In certain embodiments, Q¹ is a bond toX⁵, wherein if Q¹ is a bond to X⁵, then Y⁵ is absent.

In some embodiments, the conjugate includes at least one modified aminoacid residue of formula (IVa) or (IVb):

wherein m, R¹, R², R^(3′) R¹²′ X⁵, Y⁵′ L, W¹ and W² are as defined informula (I);

X¹ and X³ are each independently O, S or NR¹²;

Y¹, Y², Y³ and Y⁴, if present, are each independently selected fromhydrogen, halogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, amino acyl, alkylamide, substituted alkylamide, sulfonyl,thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, wherein Y¹ and Y² or Y³ andY⁴ are optionally cyclically linked;

Q¹ is a bond to either X⁴ or X⁵, wherein if Q¹ is a bond to X⁴, then X⁴is C and Y⁴ is absent, or if Q¹ is a bond to X⁵, then Y⁵ is absent; and

Q² is a bond to either X³ or X⁵, wherein if Q² is a bond to X³, then X³is NR¹² and R¹² is absent, or if Q² is a bond to X⁵, then Y⁵ is absent.

In certain embodiments, the substituents in formulae (IVa) and (IVb) areas described above for formula (I). In certain embodiments, thesubstituents m, R¹, R², R³, R¹²′ X⁵, Y⁵′ L, W¹ and W² are as defined informula (I).

In certain embodiments of formula (IVa), X¹ is O, S or NR¹². In certainembodiments of formula (IVa), X¹ is O. In certain embodiments of formula(IVa), X¹ is S. In certain embodiments of formula (IVa), X¹ is NR¹².

In certain embodiments of formula (IVb), X³ is O, S or NR¹². In certainembodiments of formula (IVb), X³ is O. In certain embodiments of formula(IVb), X³ is S. In certain embodiments of formula (IVb), X³ is NR¹².

In certain embodiments of formula (IVb), Y¹ is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y¹ is hydrogen. Incertain embodiments, Y¹ is halogen, such as F, Cl, Br or I. In certainembodiments, Y¹ is F. In certain embodiments, Y¹ is Cl. In certainembodiments, Y¹ is Br. In certain embodiments, Y¹ is I. In certainembodiments, Y¹ is alkyl or substituted alkyl. In certain embodiments,Y¹ is alkenyl or substituted alkenyl. In certain embodiments, Y¹ isalkynyl or substituted alkynyl. In certain embodiments, Y¹ is alkoxy orsubstituted alkoxy. In certain embodiments, Y¹ is amino or substitutedamino. In certain embodiments, Y¹ is carboxyl or carboxyl ester. Incertain embodiments, Y¹ is acyl or acyloxy. In certain embodiments, Y¹is acyl amino or amino acyl. In certain embodiments, Y¹ is alkylamide orsubstituted alkylamide. In certain embodiments, Y¹ is sulfonyl. Incertain embodiments, Y¹ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y¹ is aryl or substituted aryl. In certainembodiments, Y¹ is heteroaryl or substituted heteroaryl. In certainembodiments, Y¹ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y¹ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (IVb), Y² is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y² is hydrogen. Incertain embodiments, Y² is halogen, such as F, Cl, Br or I. In certainembodiments, Y² is F. In certain embodiments, Y² is Cl. In certainembodiments, Y² is Br. In certain embodiments, Y² is I. In certainembodiments, Y² is alkyl or substituted alkyl. In certain embodiments,Y² is alkenyl or substituted alkenyl. In certain embodiments, Y² isalkynyl or substituted alkynyl. In certain embodiments, Y² is alkoxy orsubstituted alkoxy. In certain embodiments, Y² is amino or substitutedamino. In certain embodiments, Y² is carboxyl or carboxyl ester. Incertain embodiments, Y² is acyl or acyloxy. In certain embodiments, Y²is acyl amino or amino acyl. In certain embodiments, Y² is alkylamide orsubstituted alkylamide. In certain embodiments, Y² is sulfonyl. Incertain embodiments, Y² is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y² is aryl or substituted aryl. In certainembodiments, Y² is heteroaryl or substituted heteroaryl. In certainembodiments, Y² is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y² is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (IVa), Y³ is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y³ is hydrogen. Incertain embodiments, Y³ is halogen, such as F, Cl, Br or I. In certainembodiments, Y³ is F. In certain embodiments, Y³ is Cl. In certainembodiments, Y³ is Br. In certain embodiments, Y³ is I. In certainembodiments, Y³ is alkyl or substituted alkyl. In certain embodiments,Y³ is alkenyl or substituted alkenyl. In certain embodiments, Y³ isalkynyl or substituted alkynyl. In certain embodiments, Y³ is alkoxy orsubstituted alkoxy. In certain embodiments, Y³ is amino or substitutedamino. In certain embodiments, Y³ is carboxyl or carboxyl ester. Incertain embodiments, Y³ is acyl or acyloxy. In certain embodiments, Y³is acyl amino or amino acyl. In certain embodiments, Y³ is alkylamide orsubstituted alkylamide. In certain embodiments, Y³ is sulfonyl. Incertain embodiments, Y³ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y³ is aryl or substituted aryl. In certainembodiments, Y³ is heteroaryl or substituted heteroaryl. In certainembodiments, Y³ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y³ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (IVa), Y⁴ (if present) is selectedfrom hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, amino acyl, alkylamide, substituted alkylamide, sulfonyl,thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl. In certain embodiments, Y⁴is hydrogen. In certain embodiments, Y⁴ is halogen, such as F, Cl, Br orI. In certain embodiments, Y⁴ is F. In certain embodiments, Y⁴ is Cl. Incertain embodiments, Y⁴ is Br. In certain embodiments, Y⁴ is I. Incertain embodiments, Y⁴ is alkyl or substituted alkyl. In certainembodiments, Y⁴ is alkenyl or substituted alkenyl. In certainembodiments, Y⁴ is alkynyl or substituted alkynyl. In certainembodiments, Y⁴ is alkoxy or substituted alkoxy. In certain embodiments,Y⁴ is amino or substituted amino. In certain embodiments, Y⁴ is carboxylor carboxyl ester. In certain embodiments, Y⁴ is acyl or acyloxy. Incertain embodiments, Y⁴ is acyl amino or amino acyl. In certainembodiments, Y⁴ is alkylamide or substituted alkylamide. In certainembodiments, Y⁴ is sulfonyl. In certain embodiments, Y⁴ is thioalkoxy orsubstituted thioalkoxy. In certain embodiments, Y⁴ is aryl orsubstituted aryl. In certain embodiments, Y⁴ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y⁴ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y⁴ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y¹ and Y² or Y³ and Y⁴ are cyclically linked toform a fused benzo ring. In certain embodiments of formula (IVa), Y³ andY⁴ are cyclically linked to form a fused benzo ring. In certainembodiments of formula (IVb), Y¹ and Y² are cyclically linked to form afused benzo ring.

In some embodiments of formula (IVa), Q¹ is a bond to X⁴ and Y⁴ isabsent.

In some embodiments of formula (IVa), Q¹ is a bond to X⁵, X⁵ is C and Y⁵is absent.

In some embodiments of formula (IVb), Q² is a bond to X³, X³ is NR¹² andR¹² is absent.

In some embodiments of formula (IVb), Q² is a bond to X⁵ and Y⁵ isabsent.

In some embodiments, R² and R³ are each independently selected fromalkyl and substituted alkyl.

In some embodiments of formulae (IVa) and (IVb), m is 1.

In some embodiments of formulae (IVa) and (IVb), R² and R³ are eachmethyl.

In some embodiments of formulae (IVa) and (IVb), or R² and R³ areoptionally cyclically linked to form a 5 or 6-membered heterocyclyl asdescribed above in formula (I).

In some embodiments of formulae (IVa) and (IVb), X¹, X³ , X⁴ and X⁵ areeach C.

In some embodiments of formulae (IVa) and (IVb), Y¹, Y² and Y³ are eachH, and one of either Y⁴ or Y⁵ is H.

In some embodiments of formulae (IVa) and (IVb), the chemical entity isa drug or a detectable label.

In some embodiments of formulae (IVa) and (IVb), W¹ is the chemicalentity, and

W² is the polypeptide.

In some embodiments of formulae (IVa) and (IVb), W¹ is the polypeptide,and W² is the chemical entity.

In some embodiments, the conjugate comprises at least one modified aminoacid residue of formula (IIIa):

In some embodiments, the conjugate comprises at least one modified aminoacid residue of formula (IIIb):

In certain embodiments, the conjugate includes at least one modifiedamino acid residue of formula (IIIc):

In some embodiments, the conjugate comprises at least one modified aminoacid residue of formula (IIId):

In some embodiments, the conjugate comprises at least one modified aminoacid residue of formula (IIIe):

In certain embodiments, the conjugate includes at least one modifiedamino acid residue of formula (IIIf):

In certain embodiments, the substituents in formulae (IIIa), (IIIb),(IIIc), (Ind), (IIe) and (IIIf) are as described above for formula(III).

Linkers Useful for Conjugates and Compounds

The present disclosure provides linkers (L) useful for the conjugatesand compounds described herein, such as conjugates and compounds of eachof the formulae disclosed herein (e.g., formulae (I), (II), (III),(IIIa), (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IVa), (IVb), (V), (VI),(VIIa), (VIIb), (VIII), (VIIIa), (VIIIb), (IX), (IXa) and (IXb) asdescribed herein). The linkers may be utilized to bind a coupling moietyto one or more moieties of interest and/or one or more polypeptides. Insome embodiments, the linker binds a coupling moiety to either apolypeptide or a chemical entity. The linker may be bound (e.g.,covalently bounded) to the coupling moiety (e.g., as described herein)at any convenient position.

In certain embodiments, the linker (L) is optional. In certainembodiments, L is not present, and thus the polypeptide or the chemicalentity is directly bonded to the coupling moiety. In certainembodiments, L is present, and thus the coupling moiety is indirectlybonded to W¹ through the linker L.

Any convenient linkers may be utilized in the subject conjugates andcompunds. In certain embodiments, L includes a group selected fromalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl amino, alkylamide, substitutedalkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl. In certain embodiments, L includes an alkyl or substitutedalkyl group. In certain embodiments, L includes an alkenyl orsubstituted alkenyl group. In certain embodiments, L includes an alkynylor substituted alkynyl group. In certain embodiments, L includes analkoxy or substituted alkoxy group. In certain embodiments, L includesan amino or substituted amino group. In certain embodiments, L includesa carboxyl or carboxyl ester group. In certain embodiments, L includesan acyl amino group. In certain embodiments, L includes an alkylamide orsubstituted alkylamide group. In certain embodiments, L includes an arylor substituted aryl group. In certain embodiments, L includes aheteroaryl or substituted heteroaryl group. In certain embodiments, Lincludes a cycloalkyl or substituted cycloalkyl group. In certainembodiments, L includes a heterocyclyl or substituted heterocyclylgroup.

In certain embodiments, L includes a polymer. For example, the polymermay include a polyalkylene glycol and derivatives thereof, includingpolyethylene glycol, methoxypolyethylene glycol, polyethylene glycolhomopolymers, polypropylene glycol homopolymers, copolymers of ethyleneglycol with propylene glycol (e.g., where the homopolymers andcopolymers are unsubstituted or substituted at one end with an alkylgroup), polyvinyl alcohol, polyvinyl ethyl ethers, polyvinylpyrrolidone,combinations thereof, and the like. In certain embodiments, the polymeris a polyalkylene glycol. In certain embodiments, the polymer is apolyethylene glycol. Other linkers are also possible, as shown in theconjugates and compounds described in more detail below.

In some embodiments, L is a linker described by the formula-(L¹)_(a)-(L²)_(b)-(L³)_(c)-(L⁴)d-(L⁵)_(e)-, wherein L¹, L², L³, L⁴ andL⁵ are each independently a linker unit, and a, b, c, d and e are eachindependently 0 or 1, wherein the sum of a, b, c, d and e is 1 to 5.

In certain embodiments, the sum of a, b, c, d and e is 1. In certainembodiments, the sum of a, b, c, d and e is 2. In certain embodiments,the sum of a, b, c, d and e is 3. In certain embodiments, the sum of a,b, c, d and e is 4. In certain embodiments, the sum of a, b, c, d and eis 5. In certain embodiments, a, b, c, d and e are each 1. In certainembodiments, a, b and c are each 1, and d and e are each 0. In certainembodiments, a and b are each 1, and c, d and e are each 0. In certainembodiments, a is 1 and b, c, d and e are each 0.

Any convenient linker units may be utilized in the subject linkers.Linker units of interest include, but are not limited to, units ofpolymers such as polyethylene glycols, polyethylenes and polyacrylates,amino acid residue(s), carbohydrate-based polymers or carbohydrateresidues and derivatives thereof, polynucleotides, alkyl groups, arylgroups, heterocycle groups, cleavable linker groups, combinationsthereof, and substituted versions thereof. In some embodiments, each ofL¹, L² , L³, L⁴ and L⁵ (if present) include one or more groupsindependently selected from a polyethylene glycol, a modifiedpolyethylene glycol, an amino acid residue, an alkyl group, asubstituted alkyl, an aryl group, a substituted aryl group, a diamine(e.g., a linking group that includes an alkylene diamine), and acleavable moiety (e.g., a chemically cleavable moiety, an enzymaticallycleavable moiety (such as, but not limited to, a protease cleavablemoiety, a glucuronidase cleavable moiety, a beta-lactamase cleavablemoiety, etc.), a photocleavable moiety, and the like).

In some embodiments, L¹ (if present) comprises a polyethylene glycol, amodified polyethylene glycol, an amino acid residue, an alkyl group, asubstituted alkyl, an aryl group, a substituted aryl group, a diamine ora cleavable moiety. In some embodiments, L¹ comprises a polyethyleneglycol. In some embodiments, L¹ comprises a modified polyethyleneglycol. In some embodiments, L¹ comprises an amino acid residue. In someembodiments, L¹ comprises an alkyl group or a substituted alkyl. In someembodiments, L¹ comprises an aryl group or a substituted aryl group. Insome embodiments, L¹ comprises a diamine. In some embodiments, L¹comprises a cleavable moiety.

In some embodiments, L² (if present) comprises a polyethylene glycol, amodified polyethylene glycol, an amino acid residue, an alkyl group, asubstituted alkyl, an aryl group, a substituted aryl group, a diamine ora cleavable moiety. In some embodiments, L² comprises a polyethyleneglycol. In some embodiments, L² comprises a modified polyethyleneglycol. In some embodiments, L² comprises an amino acid residue. In someembodiments, L² comprises an alkyl group or a substituted alkyl. In someembodiments, L² comprises an aryl group or a substituted aryl group. Insome embodiments, L² comprises a diamine. In some embodiments, L²comprises a cleavable moiety.

In some embodiments, L³ (if present) comprises a polyethylene glycol, amodified polyethylene glycol, an amino acid residue, an alkyl group, asubstituted alkyl, an aryl group, a substituted aryl group, a diamine ora cleavable moiety. In some embodiments, L³ comprises a polyethyleneglycol. In some embodiments, L³ comprises a modified polyethyleneglycol. In some embodiments, L³ comprises an amino acid residue. In someembodiments, L³ comprises an alkyl group or a substituted alkyl. In someembodiments, L³ comprises an aryl group or a substituted aryl group. Insome embodiments, L³ comprises a diamine. In some embodiments, L³comprises a cleavable moiety.

In some embodiments, L⁴ (if present) comprises a group independentlyselected from a polyethylene glycol, a modified polyethylene glycol, anamino acid residue, an alkyl group, a substituted alkyl, an aryl group,a substituted aryl group, a diamine (e.g., a linking group comprising analkylene diamine), and a cleavable moiety. In some embodiments, L⁴comprises a polyethylene glycol. In some embodiments, L⁴ comprises amodified polyethylene glycol. In some embodiments, L⁴ comprises an aminoacid residue. In some embodiments, L⁴ comprises an alkyl group or asubstituted alkyl. In some embodiments, L⁴ comprises an aryl group or asubstituted aryl group. In some embodiments, L⁴ comprises a diamine. Insome embodiments, L⁴ comprises a cleavable moiety.

In some embodiments, L⁵ (if present) comprises a group independentlyselected from a polyethylene glycol, a modified polyethylene glycol, anamino acid residue, an alkyl group, a substituted alkyl, an aryl group,a substituted aryl group, a diamine (e.g., a linking group comprising analkylene diamine), and a cleavable moiety. In some embodiments, L⁵comprises a polyethylene glycol. In some embodiments, L⁵ comprises amodified polyethylene glycol. In some embodiments, L⁵ comprises an aminoacid residue. In some embodiments, L⁵ comprises an alkyl group or asubstituted alkyl. In some embodiments, L⁵ comprises an aryl group or asubstituted aryl group. In some embodiments, L⁵ comprises a diamine. Insome embodiments, L⁵ comprises a cleavable moiety.

Any convenient cleavable moieties may be utilized as a cleavable linkerunit in the subject conjugates and compunds. In certain embodiments, thecleavable moiety is a para-amino-benzyloxycarbonyl group (PABC), ameta-amino-benzyloxycarbonyl group (MABC), a para-amino-benzyloxy group(PABO), a meta-amino-benzyloxy group (MABO), para-aminobenzyl, an acetalgroup, a disulfide, a hydrazine, a protease-cleavable moiety (e.g., aCat B cleavable moiety), a glucuronidase cleavable moiety, abeta-lactamase cleavable moiety, or an ester.

In some embodiments, L is a linker comprising-(L¹)_(a)-(L²)_(b)-(L³),-(L⁴)_(d)-(L⁵)_(e)-, where:

-(L¹)_(a)- is -(T¹-V¹)_(a)-;

-(L²)_(b)- is -(T²-V²)_(b)-;

-(L³)_(c)- is -(T³-V³)_(c)-;

-(L⁴)_(d)- is -(T⁴-V⁴)_(d)-; and

-(L⁵)_(e)- is -(T⁵-V⁵)_(e)-,

wherein T¹, T², T³, T⁴ and T⁵, if present, are tether groups;

V¹, V², V³ , Z⁴ and V⁵, if present, are covalent bonds or linkingfunctional groups; and

a, b, c, d and e are each independently 0 or 1, wherein the sum of a, b,c, d and e is 1 to 5.

Regarding the tether groups, T¹, T², T³, T⁴ and T⁵, any convenienttether groups may be utilized in the subject linkers. In someembodiments, T¹, T², T³, T⁴ and T⁵ each comprise one or more groupsindependently selected from a (C₁-C₁₂alkyl, a substituted (C₁-C₁₂)alkyl,an (EDA)_(w), (PEG), (AA)_(p), —(CR¹³OH)_(h)—, piperidin-4-amino (P4A),MABC, MABO, PABO, PABC, para-aminobenzyl, an acetal group, a disulfide,a hydrazine, a protease-cleavable moiety, a glucuronidase cleavablemoiety, a beta-lactamase cleavable moiety, and an ester, where w is aninteger from 1 to 20, n is an integer from 1 to 30, p is an integer from1 to 20, and h is an integer from 1 to 12.

In certain embodiments, when the sum of a, b, c, d and e is 2 and one ofT¹-V¹, T²-V², T³-V³, T⁴-V⁴ or T⁵-V⁵ is (PEG)_(n)-CO, then n is not 6.For example, in some instances, the linker may have the followingstructure:

where n is not 6.

In certain embodiments, when the sum of a, b, c, d and e is 2 and one ofT¹-V¹, T²-V², T³-V³, T⁴-V⁴ or T⁵-V⁵ is (C₁-C₁₂)alkyl-NR¹¹, then(C₁-C₁₂)alkyl is not a C₅-alkyl. For example, in some instances, thelinker may have the following structure:

where g is not 4.

In certain embodiments, the tether group includes an ethylene diamine(EDA) moiety, e.g., an EDA containing tether. In certain embodiments,(EDA), includes one or more EDA moieties, such as where w is an integerfrom 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1to 12 or from 1 to 6, such as 1, 2, 3, 4, 5 or 6). The linked ethylenediamine (EDA) moieties may optionally be substituted at one or moreconvenient positions with any convenient substituents, e.g., with analkyl, a substituted alkyl, an acyl, a substituted acyl, an aryl or asubstituted aryl. In certain embodiments, the EDA moiety is described bythe structure:

where q is an integer from 1 to 6 and r is 0 or 1 and each R¹² isindependently selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl. In certain embodiments, q is1, 2, 3, 4, 5 or 6. In certain embodiments, q is 1 and r is 0. Incertain embodiments, q is 1 and r is 1. In certain embodiments, q is 2and r is 0. In certain embodiments, q is 2 and r is 1. In certainembodiments, each R¹² is independently selected from hydrogen, an alkyl,a substituted alkyl, an aryl and a substituted aryl. In certainembodiments, any two adjacent R¹² groups of the EDA may be cyclicallylinked, e.g., to form a piperazinyl ring. In certain embodiments, q is 1and the two adjacent R¹² groups are an alkyl group, cyclically linked toform a piperazinyl ring. In certain embodiments, q is 1 and the adjacentR¹² groups are selected from hydrogen, an alkyl (e.g., methyl) and asubstituted alkyl (e.g., lower alkyl-OH, such as ethyl-OH or propyl-OH).

In certain embodiments, the tether group includes a piperidin-4-amino(P4A) moiety. The P4A moiety may optionally be substituted at one ormore convenient positions with any convenient substituents, e.g., withan alkyl, a substituted alkyl, a polyethylene glycol moiety, an acyl, asubstituted acyl, an aryl or a substituted aryl. In certain embodiments,the P4A moiety is described by the structure:

where R¹² is selected from hydrogen, alkyl, substituted alkyl, apolyethylene glycol moiety (e.g., a polyethylene glycol or a modifiedpolyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R¹² is a polyethylene glycol moiety. In certainembodiments, R¹² is a carboxy modified polyethylene glycol.

In certain embodiments, a tether is (PEG) where (PEG) is a polyethyleneglycol or a modified polyethylene glycol linking unit. In certainembodiments, (PEG) is described by the structure:

where n is an integer from 1 to 50, such as from 1 to 40, from 1 to 30,from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some instances, nis 3. In some instances, n is 6. In some instances, n is 12.

In certain embodiments, a tether group includes (AA)_(p), where AA is anamino acid residue. Any convenient amino acids may be utilized. Aminoacids of interest include but are not limited to, L- and D-amino acids,naturally occurring amino acids such as any of the 20 primaryalpha-amino acids and beta-alanine, non-naturally occurring amino acids(e.g., amino acid analogs), such as a non-naturally occurringalpha-amino acid or a non-naturally occurring beta-amino acid, etc. Incertain embodiments, p is 1. In certain embodiments, p is an integerfrom 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1to 12 or from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20.

In certain embodiments, a tether group includes a moiety described bythe formula —(CR¹³OH)_(h)—, where h is 0 or n is an integer from 1 to50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 orfrom 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12. Incertain embodiments, R¹³ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R¹³ is hydrogen. In certain embodiments, R¹³ isalkyl or substituted alkyl. In certain embodiments, R¹³ is alkenyl orsubstituted alkenyl. In certain embodiments, R¹³ is alkynyl orsubstituted alkynyl. In certain embodiments, R¹³ is alkoxy orsubstituted alkoxy. In certain embodiments, R¹³ is amino or substitutedamino. In certain embodiments, R¹³ is carboxyl or carboxyl ester. Incertain embodiments, R¹³ is acyl or acyloxy. In certain embodiments, R¹³is acyl amino or amino acyl. In certain embodiments, R¹³ is alkylamideor substituted alkylamide. In certain embodiments, R¹³ is sulfonyl. Incertain embodiments, R¹³ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R¹³ is aryl or substituted aryl. In certainembodiments, R¹³ is heteroaryl or substituted heteroaryl. In certainembodiments, R¹³ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R¹³ is heterocyclyl or substituted heterocyclyl. In certainembodiments, R¹³ is selected from hydrogen, an alkyl, a substitutedalkyl, an aryl, and a substituted aryl.

Regarding V¹, V², V³, V⁴ and V⁵, any convenient linking functionalgroups may be utilized in the subject linkers. Linking functional groupsof interest include, but are not limited to, amino, carbonyl, amido,oxycarbonyl, carboxy, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl,thio, oxy, phospho, phosphoramidate, thiophosphoraidate, and the like.In some embodiments, V¹, V², V³, V⁴ and V⁵ are each independentlyselected from the group consisting of a covalent bond, —CO—, —NR¹¹—,—CONR¹¹—, —NR¹¹CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO₂—,—SO₂NR¹¹—, —NR¹¹SO₂— and —P(O)OH—. In some embodiment, R¹¹ is selectedfrom hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, R¹¹ is hydrogen. Incertain embodiments, R¹¹ is alkyl or substituted alkyl. In certainembodiments, R¹¹ is alkenyl or substituted alkenyl. In certainembodiments, R¹¹ is alkynyl or substituted alkynyl. In certainembodiments, R¹¹ is alkoxy or substituted alkoxy. In certainembodiments, R¹¹ is carboxyl or carboxyl ester. In certain embodiments,R¹¹ is acyl or acyloxy. In certain embodiments, R¹¹ is acyl amino oramino acyl. In certain embodiments, R¹¹ is alkylamide or substitutedalkylamide. In certain embodiments, R¹¹ is sulfonyl. In certainembodiments, R¹¹ is thioalkoxy or substituted thioalkoxy. In certainembodiments, R¹¹ is aryl or substituted aryl. In certain embodiments,R¹¹ is heteroaryl or substituted heteroaryl. In certain embodiments, R¹¹is cycloalkyl or substituted cycloalkyl. In certain embodiments, R¹¹ isheterocyclyl or substituted heterocyclyl.

In some embodiments, a tether includes a MABC group described by thefollowing structure:

In some embodiments, a tether includes a MABO group described by thefollowing structure:

In some embodiments of the MABO and MABC tether structures shown above,R is selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl. In some embodiments of theMABO and MABC tether structures shown above, R is a carbohydrate orcarbohydrate derivative.

In some embodiments, a tether includes a PABC group described by thefollowing structure:

In some embodiments, a tether includes a PABO group described by thefollowing structure:

In some embodiments, a tether includes a para-aminobenzyl (PAB) groupdescribed by the following structure:

In some embodiments of the PABO, PABC, MABO, MABC and PAB tetherstructures shown above, R¹² is selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments of PABO, PABC, MABO, MABC and PAB, R¹² is selectedfrom hydrogen, an alkyl, a substituted alkyl, an aryl and a substitutedaryl. In certain embodiments of PABO, PABC, MABO, MABC and PAB, R¹² isselected from hydrogen, an alkyl (e.g., methyl) and a substituted alkyl(e.g., lower alkyl-OH, such as ethyl-OH or propyl-OH). In someembodiments, any of the PABO, PABC, MABO, MABC and PAB tether structuresshown above may be further substituted with one or more convenient aryland/or alkyl substituents. In certain embodiments of PABO, PABC, MABO,MABC and PAB, R¹² is hydrogen. The divalent PABO, PABC, MABO, MABC andPAB tether groups may be covalently bound to adjacent moieties via anyconvenient chemistries.

In certain embodiments, the tether group includes an acetal group, adisulfide, a hydrazine, a glucuronidase cleavable moiety, abeta-lactamase cleavable moiety, or an ester. In some embodiments, thetether group is an acetal group. In some embodiments, the tether groupis a disulfide. In some embodiments, the tether group is a hydrazine. Insome embodiments, the tether group is a glucuronidase cleavable moiety.In some embodiments, the tether group is a beta-lactamase cleavablemoiety. In some embodiments, the tether group is an ester.

In some embodiments, in the subject linker:

T¹ is selected from a (C₁-C₁₂)alkyl and a substituted (C₁ ⁻C₁₂)alkyl;

T², T³, T⁴ and T⁵ are each independently selected from (EDA)_(w), (PEG),a (C₁-C₁₂)alkyl, a substituted (C₁-C₁₂)alkyl, (AA)_(p), —(CR¹³OH)_(h)—,piperidin-4-amino (P4A), a MABC, a MABO, a PABO, a PABC,para-aminobenzyl, an acetal group, a disulfide, a hydrazine, aprotease-cleavable moiety, a glucuronidase cleavable moiety, abeta-lactamase cleavable moiety, an ester, (AA)_(p)-MABC-(AA),(AA)_(p)-MABO-(AA)_(p), (AA)_(p)-PABO-(AA)_(p) and(AA)_(p)-PABC-(AA)_(p); and

V¹, V², V³, V⁴ and V⁵ are each independently selected from the groupconsisting of: a covalent bond, —CO—, —NR¹¹—, —CONR¹¹—, —NR¹¹CO—,—C(O)O—, —OC(O)—, —O—, —S—, -S(O)—, —SO₂—, —SO₂NR¹¹—, —NR¹¹SO₂—, and—P(O)OH—;

wherein:

(PEG)_(n) is

where n is an integer from 1 to 30;

EDA is an ethylene diamine moiety having the following structure:

where q is an integer from 1 to 6 and r is 0 or 1;

piperidin-4-amino (P4A) is

AA is an amino acid residue, where p is an integer from 1 to 20;

PABC is para-amino-benzyloxycarbonyl, MABC ismeta-amino-benzyloxycarbonyl, PABO is para-amino-benzyloxy, and MABO ismeta-amino-benzyloxy;

each R¹¹ and R¹² is independently selected from hydrogen, an alkyl, asubstituted alkyl, a PEG, an aryl and a substituted aryl, wherein anytwo adjacent R¹² groups may be cyclically linked to form a piperazinylring; and

R¹³ is selected from hydrogen, an alkyl, a substituted alkyl, an aryl,and a substituted aryl.

In certain embodiments, T¹, T², T³, T⁴ and T⁵ and V¹, V², V³, V⁴ and V⁵are selected from the following table, e.g., one row of the followingtable:

T¹ V¹ T² V² T³ V³ T⁴ V⁴ T⁵ V⁵ (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— — —— — — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— — — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) — — — — — — — (C₁-C₁₂)alkyl —CONR¹¹—(PEG)_(n) —NR¹¹— — — — — — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹—(PEG)_(n) —NR¹¹— — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— — — — — — —(C₁-C₁₂)alkyl —CONR¹¹— (C₁-C₁₂)alkyl —NR¹¹— — — — — — — (C₁-C₁₂)alkyl—CONR¹¹— (PEG)_(n) —CO— (EDA)_(w) — — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w)— — — — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— (CR¹³OH)_(h) —CONR¹¹—(C₁-C₁₂)alkyl —CO— — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (C₁-C₁₂)alkyl—CO— — — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — — — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — MABO — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABO — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABC — — —(C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— (CR¹³OH)_(h) —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (C₁-C₁₂)alkyl —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (C₁-C₁₂)alkyl —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p)- — — — PABC-(AA)_(p) (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p)- —PABC- — (AA)_(p) (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO—(AA)_(p)- — — — PABO — — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n)—CO— (AA)_(p) — PABO — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n)—SO_(2—) (AA)_(p) — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) —PABC- — — — (AA)_(p) (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) —PABC — (AA)_(p) — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— (CR¹³OH)_(h)—CONR¹¹— (PEG)_(n) —CO— — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n)—CO— MABC- — — — (AA)_(p)- — — — (C₁-C₁₂)alkyl —CO— (AA)p —NR¹¹—(PEG)_(n) —CO— MABC — (AA)_(p) — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO—MABO — — — — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— MABO —— — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— PABO — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— PABC — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— MABC — (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (CR¹³OH)_(h) —CO— — — — — — — (C₁-C₁₂)alkyl —CONR¹¹—substituted —NR¹¹— (PEG)_(n) —CO— — — — — (C₁-C₁₂)alkyl (C₁-C₁₂)alkyl—SO₂— (C₁-C₁₂)alkyl —CO— — — — — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n)—CO— (AA)p — PABC —NR¹¹— — — (C₁-C₁₂)alkyl —CONR¹¹— (C₁-C₁₂)alkyl —(CR¹³OH)_(h) —CONR¹¹— — — — — (C₁-C₁₂)alkyl —CO— P4A —CO— (C₁-C₁₂)alkyl—CO— (AA)_(p) — PABO —CO— (C₁-C₁₂)alkyl —CO— P4A —CO— (C₁-C₁₂)alkyl —CO—(AA)_(p) — PABO — (C₁-C₁₂)alkyl —CO— P4A —CO— (C₁-C₁₂)alkyl —CO—(AA)_(p) — PABC- — (AA)_(p) (C₁-C₁₂)alkyl —CO— P4A —CO— (C₁-C₁₂)alkyl—CO— (AA)_(p) — — —

In some embodiments, L is a linker comprising-(L¹)_(a)-(L²)_(b)-(L³)_(c)-(L⁴)_(d)-(L⁵)_(e), where -(L¹)_(a)is-(T¹V¹)_(a)-; -(L²)_(b)- is -(T²-V²)_(b)-; -(L³)_(c)- is -(T³-V³)_(c)-;-(L⁴)_(d)- is -(T⁴-V⁴)_(d)-; and -(L⁵)_(e)- is -(T⁵-V⁵)_(e)-. In certainembodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹¹—, T² is (PEG)_(n), V² is—CO—, T³ is absent, V³ is absent, T⁴ is absent, V⁴ is absent, T⁵ isabsent and V⁵ is absent. In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹is —CO—, T² is (AA)_(p), V² is —NR¹¹—, T³ is (PEG)_(n), V³ is —CO—, T⁴is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent. In certainembodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p), V² isabsent, T³ is absent , V³ is absent , T is absent, V⁴ is absent, T⁵ isabsent and V⁵ is absent. In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹is —CONR¹¹—, T² is (PEG)_(n), V² is —NR¹¹—, T³ is absent, V³ is absent,T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent. In certainembodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p), V² is—NR¹¹—, T³ is (PEG)_(n), V³ is —NR¹¹—, T⁴ is absent, V⁴ is absent, T⁵ isabsent and V⁵ is absent. In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹is —CO—, T² is (EDA)_(w), V² is —CO—, T³ is absent, V³ is absent, T⁴ isabsent, V⁴ is absent, T⁵ is absent and V⁵ is absent. In certainembodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹¹—, T² is (C₁-C₁₂)alkyl,V² is —NR¹¹—, T³ is absent, V³ is absent, T⁴ is absent, V⁴ is absent, T⁵is absent and V⁵ is absent. In certain embodiments, T¹ is (C₁-C₁₂)alkyl,V¹ is —CONR¹¹—, T² is (PEG)_(n), V² is —CO—, T³ is (EDA)_(w), V³ isabsent, T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent. Incertain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (EDA)_(w),V² is absent, T³ is absent, V³ is absent, T⁴ is absent, V⁴ is absent, T⁵is absent and V⁵ is absent. In certain embodiments, T¹ is (C₁-C₁₂)alkyl,V¹ is —CO—, T² is (EDA),, V² is —CO—, T³ is (CR¹³OH)_(h), V³ is—CONR¹¹—, T⁴ is (C₁-C₁₂)alkyl, V⁴ is —CO—, T⁵ is absent and V⁵ isabsent. In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is(AA)_(p), V² is —NR¹¹—, T³ is (C₁-C₁₂)alkyl, V³ is —CO—, T⁴ is absent,V⁴ is absent, T⁵ is absent and V⁵ is absent. In certain embodiments, T¹is (C₁-C₁₂)alkyl, V¹ is —CONR¹¹—, T² is (PEG)_(n), V² is —CO—, T³ is(AA)_(p), V³ is absent, T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵is absent. In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T²is (EDA)_(w), V² is —CO—, T³ is (CR¹³OH)_(h), V³ is —CO—, T⁴ is(AA)_(p), V⁴ is absent, T⁵ is absent and V⁵ is absent. In certainembodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p), V² is—NR¹¹—, T³ is (C₁-C₁₂)alkyl, V³ is —CO—, T⁴ is (AA)_(p), V⁴ is absent,T⁵ is absent and V⁵ is absent. In certain embodiments, T¹ is(C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p), V² is —NR¹¹—, T³ is (PEG), V³is —CO—, T⁴ is (AA)_(p), V⁴ is absent, T⁵ is absent and V⁵ is absent. Incertain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p), V²is —NR¹¹—, T³ is (C₁-C₁₂)alkyl, V³ is —CO—, T⁴ is (AA)_(p), V⁴ isabsent, T⁵ is absent and V⁵ is absent. In certain embodiments, T¹ is(C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p), V² is —NR¹¹—, T³ is(PEG)_(n), V³ is —CO—, T⁴ is (AA)_(p)-PABC-(AA)_(p) , where each p isindependently 0, 1, 2, 3, 4 , 5 or 6, V⁴ is absent, T⁵ is absent and V⁵is absent. In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T²is (AA)_(p), V² is —NR¹¹—, T³ is (PEG), V³ is —CO—, T⁴ is (AA)_(p)-PABOwhere p is 0, 1, 2, 3, 4 , 5 or 6, V⁴ is absent, T⁵ is absent and V⁵ isabsent. In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is(AA)_(p), V² is —NR¹¹—, T³ is (PEG), V³ is —SO₂—, T⁴ is (AA)_(p), V⁴ isabsent, T⁵ is absent and V⁵ is absent. In certain embodiments, T¹ is(C₁-C₁₂)alkyl, V¹ is —CONR¹¹—, T² is (PEG), V² is —CO—, T³ is (AA)_(p),V³ is absent, T⁴ is PABC-(AA)_(p) where p is 0, 1, 2, 3, 4, 5 or 6, V⁴is absent, T⁵ is absent and V⁵ is absent. In certain embodiments, T¹ is(C₁-C₁₂)alkyl, V¹ is —CO—, T² is (EDA)_(w), V² is —CO—, T³ is(CR¹³OH)_(h), V³ is —CONR¹¹—, T⁴ is (PEG), V⁴ is —CO—, T⁵ is absent andV⁵ is absent. In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—,T² is (AA)_(p), V² is —NR¹¹—, T³ is (PEG)_(n), V³ is —CO—, T⁴ isMABC-(AA)_(p)- where p is 0, 1, 2, 3, 4, 5 or 6, V⁴ is absent, T⁵ isabsent and V⁵ is absent. In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹is —CONR¹¹—, T² is (PEG), V² is —CO—, T³ is MABO, V³ is absent, T⁴ isabsent, V⁴ is absent, T⁵ is absent and V⁵ is absent. In certainembodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p), V² is—NR¹¹—, T³ is (PEG), V³ is —CO—, T⁴ is MABO, V⁴ is absent, T⁵ is absentand V⁵ is absent. In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is—CONR¹¹—, T² is (PEG), V² is —CO—, T³ is MABC, V³ is absent, T⁴ is(AA)_(p), V⁴ is absent, T⁵ is absent and V⁵ is absent. In certainembodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (CR¹³OH)_(h), V² is—CO—, T³ is absent, V³ is absent, T⁴ is absent, V⁴ is absent, T⁵ isabsent and V⁵ is absent. In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹is —CONR¹¹—, T² is substituted (C₁-C₁₂)alkyl, V² is —NR¹¹—, T³ is (PEG),V³ is —CO—, T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent.In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —SO₂-, T² is(C₁-C₁₂)alkyl, V² is —CO—, T³ is absent, V³ is absent, T⁴ is absent, V⁴is absent, T⁵ is absent and V⁵ is absent. In certain embodiments, T¹ is(C₁-C₁₂)alkyl, V¹ is —CONR¹¹—, T² is (PEG), V² is —CO—, T³ is (AA)_(p),V³ is absent, T⁴ is PABC, V⁴ is —NR¹¹—, T⁵ is absent and V⁵ is absent.In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹¹—, T² is(C₁-C₁₂)alkyl, V² is absent, T³ is (CR¹³OH)_(h), V³ is —CONR¹¹—, T⁴ isabsent, V⁴ is absent, T⁵ is absent and V⁵ is absent. In certainembodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is P4A, V² is —CO—, T³is (C₁-C₁₂)alkyl, V³ is —CO—, T⁴ is (AA)_(p), V⁴ is absent, T⁵ is absentand V⁵ is absent. In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is—CONR¹¹—, T² is (PEG), V² is —CO—, T³ is (AA)_(p), V³ is absent, T⁴ isMABO, V⁴ is absent, T⁵ is absent and V⁵ is absent. In certainembodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹¹—, T² is (PEG), V² is—CO—, T³ is (AA)_(p), V³ is absent, T⁴ is PABO, V⁴ is absent, T⁵ isabsent and V⁵ is absent. In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹is CONR¹¹—, T² is (PEG), V² is —CO—, T³ is (AA)_(p), V³ is absent, T⁴ isPABC, V⁴ is absent, T⁵ is absent and V⁵ is absent. In certainembodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p), V² is—NR¹¹—, T³ is (PEG), V³ is —CO—, T⁴ is PABO, V⁴ is absent, T⁵ is absentand V⁵ is absent. In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is—CO—, T² is (AA)_(p), V² is —NR¹¹—, T³ is (PEG)_(n), V³ is —CO—, T⁴ isPABC, V⁴ is absent, T⁵ is absent and V⁵ is absent.

In some embodiments, L is a linker comprising-(L¹)_(a)-(L²)_(b)-(L³)_(c)-(L⁴)_(d)-(L⁵)_(e)-, where -(L¹)_(a)- is-(T¹-V¹)_(a)-; -(L²)_(b)- is -(T²-V²)_(b)-; -(L³)_(e)- is -(T³-V³),-;-(L⁴)_(d)- is -(T⁴-V⁴)_(d)-; and -(L⁵)_(e)- is -(T⁵-V⁵)_(e)-. In certainembodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p), V² is—NR¹¹—, T³ is (PEG), V³ is —CO—, T⁴ is (AA)p, V⁴ is absent, T⁵ isPABC-(AA)p, and V⁵ is absent. In certain embodiments, T¹ is(C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p), V² is —NR¹¹—, T³ is(PEG)_(n), V³ is —CO—, T⁴ is (AA)p, V⁴ is absent, T⁵ is PABO, and V⁵ isabsent. In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is(PEG), V² is —CO—, T³ is (AA)p, V³ is absent, T⁴ is PABC, V⁴ is absent,T⁵ is (AA)p, and V⁵ is absent. In certain embodiments, T¹ is(C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p), V² is —NR¹¹—, T³ is (PEG), V³is —CO—, T⁴ is MABC, V⁴ is absent, T⁵ is (AA)p, and V⁵ is absent. Incertain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is P4A, V² is—CO—, T³ is (C₁-C₁₂)alkyl, V³ is —CO—, T⁴ is (AA)p, V⁴ is absent, T⁵ isPABO, and V⁵ is —CO—. In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is—CO—, T² is P4A, V² is —CO—, T³ is (C₁-C₁₂)alkyl, V³ is —CO—, T⁴ is(AA)p, V⁴ is absent, T⁵ is PABO, and V⁵ is absent. In certainembodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is P4A, V² is —CO—, T³is (C₁-C₁₂)alkyl, V³ is —CO—, T⁴ is (AA)p, V⁴ is absent, T⁵ isPABC-(AA)p, and V⁵ is absent. In certain embodiments, T¹ is(C₁-C₁₂)alkyl, V¹ is —CO—, T² is P4A, V² is —CO—, T³ is (C₁-C₁₂)alkyl,V³ is —CO—, T⁴ is (AA)_(p), V⁴ is absent, T⁵ is absent and V⁵ is absent.

In some embodiments, the linker is described by one of the followingstructures:

In certain embodiments of the linker structures depicted above, each fis independently 0 or an integer from 1 to 12; each w is independently 0or an integer from 1 to 20; each n is independently 0 or an integer from1 to 30; each p is independently 0 or an integer from 1 to 20; each h isindependently 0 or an integer from 1 to 12; each R is independentlyhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl; and each R′ is independently H, a sidechain ofan amino acid, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments of the linkerstructures depicted above, each f is independently 0, 1, 2, 3, 4, 5 or6; each w is independently 0, 1, 2, 3, 4, 5 or 6; each n isindependently 0, 1, 2, 3, 4, 5 or 6; each p is independently 0, 1, 2, 3,4, 5 or 6; and each h is independently 0, 1, 2, 3, 4, 5 or 6. In certainembodiments of the linker structures depicted above, each R isindependently H, methyl or —(CH₂)_(m)—OH where m is 1, 2, 3 or 4 (e.g.,2).

In certain embodiments, the linker includes a cleavable group, e.g., asdescribed in the following structures:

In certain embodiments of the linker structures depicted above, each fis independently 0 or an integer from 1 to 12; each w is independently 0or an integer from 1 to 20; each n is independently 0 or an integer from1 to 30; each p is independently 0 or an integer from 1 to 20; each h isindependently 0 or an integer from 1 to 12; each R is independentlyhydrogen, alkyl, substituted alkyl, a polyethylene glycol moiety,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; andeach R′ is independently H, a sidechain of an amino acid, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments of the linker structures depicted above, each f isindependently 0, 1, 2, 3, 4, 5 or 6; each w is independently 0, 1, 2, 3,4, 5 or 6; each n is independently 0, 1, 2, 3, 4, 5 or 6; each p isindependently 0, 1, 2, 3, 4, 5 or 6; and each h is independently 0, 1,2, 3, 4, 5 or 6. In certain embodiments of the linker structuresdepicted above, each R is independently H, methyl or —(CH₂)_(m)—OH wherem is 1, 2, 3 or 4 (e.g., 2).

Compounds Useful for Producing Conjugates

The present disclosure provides hydrazinyl-pyrrolo compounds useful forproducing the conjugates described herein. In certain embodiments, thehydrazinyl-pyrrolo compound may be a coupling moiety useful forconjugation of a polypeptide and a second moiety. For example, thehydrazinyl-pyrrolo compound may be bound to the polypeptide and alsobound to the second moiety, thus indirectly binding the polypeptide andthe second moiety together.

In certain instances, the hydrazinyl-pyrrolo compound may be a compoundof formula (V):

wherein

one of Q³ and Q⁴ is (CH₂)_(m)NR³NHR² and the other is Y⁴;

m is 0 or 1;

R² and R³ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, orR² and R³ are optionally cyclically linked to form a 5 or 6-memberedheterocyclyl;

X¹, X², X³ and X⁴ are each independently selected from C, N, O and S,wherein one of X¹, X², X³ and X⁴ is optional;

Y¹, Y², Y³ and Y⁴ are each independently selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl, wherein Y¹ and Y² or Y² and Y³ are optionallycyclically linked, and wherein when Q⁴ is Y⁴, then Y³ and Y⁴ areoptionally cyclically linked;

one of R¹⁶, Y¹, Y², Y³ or Q⁴ is -L-W¹, wherein if Q⁴ is -L-W¹, then Q³is —(CH₂)_(m)NR³NHR² and Y⁴ is absent; and wherein if one of Y¹, Y², Y³or Q⁴ is -L-W¹, then R¹⁶ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

L is an optional linker (e.g., a linker as described herein); and

W¹ is selected from a polypeptide and a chemical entity.

For example, L may be a linker such as where:

L is a linker comprising-(T¹-V¹)_(a)-(T²-V²)_(b)-(T³-V³),-(T⁴-V⁴)_(d)-(T⁵-V⁵),-, where a, b, c,d and e are each independently 0 or 1, where the sum of a, b, c, d and eis 1 to 5;

T¹, T², T³, T⁴ and T⁵ are each independently selected from(C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, (EDA)_(w), (PEG)_(n),(AA)_(p), —(CR¹³OH)_(h)—, piperidin-4-amino (P4A),para-amino-benzyloxycarbonyl (PABC), a meta-amino-benzyloxycarbonyl(MABC), a para-amino-benzyloxy (PABO), a meta-amino-benzyloxy (MABO),para-aminobenzyl, an acetal group, a disulfide, a hydrazine, aprotease-cleavable moiety, a glucuronidase cleavable moiety, abeta-lactamase cleavable moiety, and an ester, where EDA is an ethylenediamine moiety, PEG is a polyethylene glycol or a modified polyethyleneglycol, and AA is an amino acid residue;

w is an integer from 1 to 20;

n is an integer from 1 to 30;

p is an integer from 1 to 20;

h is an integer from 1 to 12; and

V¹, V², V³, V⁴ and V⁵ are each independently selected from the groupconsisting of a covalent bond, —CO—, —NR¹¹—, —CONR¹¹—, —NR¹¹CO—,—C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO₂—, —SO₂NR¹¹—, —NR¹¹SO₂— and—P(O)OH—.

In certain embodiments, one of Q³ and Q⁴ is (CH₂)_(m)NR³NHR² and theother is Y⁴. In certain embodiments, Q³ is (CH₂)NR³NHR² and Q⁴ is Y⁴. Incertain embodiments, Q⁴ is —(CH₂)NR³NHR² and Q³ is Y⁴. In certainembodiments, m is 0 or 1. In certain embodiments, m is 0. In certainembodiments, m is 1.

In certain embodiments, R² is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R² is hydrogen. In certain embodiments, R² is alkylor substituted alkyl. In certain embodiments, R² is alkenyl orsubstituted alkenyl. In certain embodiments, R² is alkynyl orsubstituted alkynyl. In certain embodiments, R² is alkoxy or substitutedalkoxy. In certain embodiments, R² is amino or substituted amino. Incertain embodiments, R² is carboxyl or carboxyl ester. In certainembodiments, R² is acyl or acyloxy. In certain embodiments, R² is acylamino or amino acyl. In certain embodiments, R² is alkylamide orsubstituted alkylamide. In certain embodiments, R² is sulfonyl. Incertain embodiments, R² is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R² is aryl or substituted aryl. In certainembodiments, R² is heteroaryl or substituted heteroaryl. In certainembodiments, R² is cycloalkyl or substituted cycloalkyl. In certainembodiments, R² is heterocyclyl or substituted heterocyclyl.

In certain embodiments, R² is alkyl or substituted alkyl. For example,R² may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R² is methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R² ismethyl.

In certain embodiments, R³ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R³ is hydrogen. In certain embodiments, R³ is alkylor substituted alkyl. In certain embodiments, R³ is alkenyl orsubstituted alkenyl. In certain embodiments, R³ is alkynyl orsubstituted alkynyl. In certain embodiments, R³ is alkoxy or substitutedalkoxy. In certain embodiments, R³ is amino or substituted amino. Incertain embodiments, R³ is carboxyl or carboxyl ester. In certainembodiments, R³ is acyl or acyloxy. In certain embodiments, R³ is acylamino or amino acyl. In certain embodiments, R³ is alkylamide orsubstituted alkylamide. In certain embodiments, R³ is sulfonyl. Incertain embodiments, R³ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R³ is aryl or substituted aryl. In certainembodiments, R³ is heteroaryl or substituted heteroaryl. In certainembodiments, R³ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R³ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, R³ is alkyl or substituted alkyl. For example,R³ may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R³ is methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R³ ismethyl.

In certain embodiments, R² and R³ are each independently selected fromalkyl and substituted alkyl. For example, R² may be alkyl or substitutedalkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆alkyl or C₁-C₆ substituted alkyl), and R³ may be alkyl or substitutedalkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆alkyl or C₁-C₆ substituted alkyl). In some cases, R² and R³ are eachindependently selected from methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R²and R³ are each methyl.

In certain embodiments, R² and R³ are optionally cyclically linked toform a 5 or 6-membered heterocyclyl. In some instances, R² and R³(together with the atoms to which they are attached) may be cyclicallylinked to form a 5-membered heterocyclyl. In some instances, R² and R³(together with the atoms to which they are attached) may be cyclicallylinked to form a 6-membered heterocyclyl. For example, R² and R³ mayeach independently be an alkyl or substituted alkyl, such as, C₁-C_(1o)alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆substituted alkyl), where R² and R³ are optionally cyclically linked toform a 5 or 6-membered heterocyclyl, as described above. In someinstances, one or more carbon atoms in R² and/or R³ may be replaced witha heteroatom, such as N, O, or S.

In certain embodiments, X¹ is selected from C, N, O and S. In certainembodiments, X¹ is C. In certain embodiments, X¹ is N. In certainembodiments, X¹ is O. In certain embodiments, X¹ is S. In certainembodiments, X¹ is absent.

In certain embodiments, X² is selected from C, N, O and S. In certainembodiments, X² is C. In certain embodiments, X² is N. In certainembodiments, X² is O. In certain embodiments, X² is S. In certainembodiments, X² is absent.

In certain embodiments, X³ is selected from C, N, O and S. In certainembodiments, X³ is C. In certain embodiments, X³ is N. In certainembodiments, X³ is O. In certain embodiments, X³ is S. In certainembodiments, X³ is absent.

In certain embodiments, X⁴ is selected from C, N, O and S. In certainembodiments, X⁴ is C. In certain embodiments, X⁴ is N. In certainembodiments, X⁴ is O. In certain embodiments, X⁴ is S. In certainembodiments, X⁴ is absent.

Various combinations of X¹, X², X³ and X⁴ are possible. For example, incertain embodiments, each of X¹, X², X³ and X⁴ is C. In other instances,three of X¹, X², X³ and X⁴ are C and one of X¹, X², X³ and X⁴ is N. Inother embodiments, two of X¹, X², X³ and X⁴ are C and two of X¹, X², X³and X⁴ are N. In other embodiments, one of X¹, X², X³ and X⁴ is C andthree of X¹, X², X³ and X⁴ is are N. In other embodiments, one of X¹,X², X³ and X⁴ is absent, two of X¹, X², X³ and X⁴ are C and one of X¹,X², X³ and X⁴ is N. In other embodiments, one of X¹, X², X³ and X⁴ isabsent, two of X¹, X², X³ and X⁴ are C and one of X¹, X², X³ and X⁴ isS. In other embodiments, one of X¹, X², X³ and X⁴ is absent, two of X¹,X², X³ and X⁴ are C and one of X¹, X², X³ and X⁴ is O. Othercombinations of C, N, O and S are possible for X¹, X², X³ and X⁴ asdesired.

In certain embodiments, Y¹ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y¹ is hydrogen. In certain embodiments, Y¹ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y¹ is F. Incertain embodiments, Y¹ is Cl. In certain embodiments, Y¹ is Br. Incertain embodiments, Y¹ is I. In certain embodiments, Y¹ is alkyl orsubstituted alkyl. In certain embodiments, Y¹ is alkenyl or substitutedalkenyl. In certain embodiments, Y¹ is alkynyl or substituted alkynyl.In certain embodiments, Y¹ is alkoxy or substituted alkoxy. In certainembodiments, Y¹ is amino or substituted amino. In certain embodiments,Y¹ is carboxyl or carboxyl ester. In certain embodiments, Y¹ is acyl oracyloxy. In certain embodiments, Y¹ is acyl amino or amino acyl. Incertain embodiments, Y¹ is alkylamide or substituted alkylamide. Incertain embodiments, Y¹ is sulfonyl. In certain embodiments, Y¹ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y¹ is arylor substituted aryl. In certain embodiments, Y¹ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y¹ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y¹ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y² is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y² is hydrogen. In certain embodiments, Y² ishalogen, such as F, Cl, Br or I. In certain embodiments, Y² is F. Incertain embodiments, Y² is Cl. In certain embodiments, Y² is Br. Incertain embodiments, Y² is I. In certain embodiments, Y² is alkyl orsubstituted alkyl. In certain embodiments, Y² is alkenyl or substitutedalkenyl. In certain embodiments, Y² is alkynyl or substituted alkynyl.In certain embodiments, Y² is alkoxy or substituted alkoxy. In certainembodiments, Y² is amino or substituted amino. In certain embodiments,Y² is carboxyl or carboxyl ester. In certain embodiments, Y² is acyl oracyloxy. In certain embodiments, Y² is acyl amino or amino acyl. Incertain embodiments, Y² is alkylamide or substituted alkylamide. Incertain embodiments, Y² is sulfonyl. In certain embodiments, Y² isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y² is arylor substituted aryl. In certain embodiments, Y² is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y² is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y² is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y³ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y³ is hydrogen. In certain embodiments, Y³ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y³ is F. Incertain embodiments, Y³ is Cl. In certain embodiments, Y³ is Br. Incertain embodiments, Y³ is I. In certain embodiments, Y³ is alkyl orsubstituted alkyl. In certain embodiments, Y³ is alkenyl or substitutedalkenyl. In certain embodiments, Y³ is alkynyl or substituted alkynyl.In certain embodiments, Y³ is alkoxy or substituted alkoxy. In certainembodiments, Y³ is amino or substituted amino. In certain embodiments,Y³ is carboxyl or carboxyl ester. In certain embodiments, Y³ is acyl oracyloxy. In certain embodiments, Y³ is acyl amino or amino acyl. Incertain embodiments, Y³ is alkylamide or substituted alkylamide. Incertain embodiments, Y³ is sulfonyl. In certain embodiments, Y³ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y³ is arylor substituted aryl. In certain embodiments, Y³ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y³ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y³ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y⁴ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y⁴ is hydrogen. In certain embodiments, Y⁴ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y⁴ is F. Incertain embodiments, Y⁴ is Cl. In certain embodiments, Y⁴ is Br. Incertain embodiments, Y⁴ is I. In certain embodiments, Y⁴ is alkyl orsubstituted alkyl. In certain embodiments, Y⁴ is alkenyl or substitutedalkenyl. In certain embodiments, Y⁴ is alkynyl or substituted alkynyl.In certain embodiments, Y⁴ is alkoxy or substituted alkoxy. In certainembodiments, Y⁴ is amino or substituted amino. In certain embodiments,Y⁴ is carboxyl or carboxyl ester. In certain embodiments, Y⁴ is acyl oracyloxy. In certain embodiments, Y⁴ is acyl amino or amino acyl. Incertain embodiments, Y⁴ is alkylamide or substituted alkylamide. Incertain embodiments, Y⁴ is sulfonyl. In certain embodiments, Y⁴ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y⁴ is arylor substituted aryl. In certain embodiments, Y⁴ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y⁴ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y⁴ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y¹ and Y² or Y² and Y³ are cyclically linked toform a fused benzo ring. In certain embodiments, Y¹ and Y² arecyclically linked to form a fused benzo ring. In certain embodiments, Y²and Y³ are cyclically linked to form a fused benzo ring. In certainembodiments, when Q⁴ is Y⁴, then Y³ and Y⁴ are optionally cyclicallylinked.

In certain embodiments, one of R¹⁶, Y¹, Y², Y³ or Q⁴ is -L-W¹, whereinif Q⁴ is -L-W¹, then Q³ is —(CH₂)_(m)NR³NHR² and Y⁴ is absent. Incertain embodiments, R¹⁶ is -L-W¹. In certain embodiments, Y¹ is -L-W¹.In certain embodiments, Y² is -L-W¹. In certain embodiments, Y³ is-L-W¹. In certain embodiments, Q⁴ is -L-W¹. In certain embodiments, ifQ⁴ is -L-W¹, then Q³ is —(CH₂)_(m)NR³NHR² and Y⁴ is absent.

In certain embodiments, if one of Y¹, Y², Y³ or Q⁴ is -L-W¹, then R¹⁶ isis not -L-W¹. In these embodiments, R¹⁶ is selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In some instances, R¹⁶ is hydrogen. In someinstances, R¹⁶ is alkyl or substituted alkyl. In some instances, R¹⁶ isalkenyl or substituted alkenyl. In some instances, R¹⁶ is alkynyl orsubstituted alkynyl. In some instances, R¹⁶ is aryl or substituted aryl.In some instances, R¹⁶ is heteroaryl or substituted heteroaryl. In someinstances, R¹⁶ is cycloalkyl or substituted cycloalkyl. In someinstances, R¹⁶ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, L is an optional linker. In certain embodiments,L is not present, and thus the nitrogen of the indole ring is directlybonded to W¹. In certain embodiments, L is present, and thus thenitrogen of the indole ring is indirectly bonded to W¹ through thelinker L. Further description of the linker, L, is found in thedisclosure herein.

For instance, in certain embodiments, L includes a group selected fromalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl amino, alkylamide, substitutedalkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl. In certain embodiments, L includes an alkyl or substitutedalkyl group. In certain embodiments, L includes an alkenyl orsubstituted alkenyl group. In certain embodiments, L includes an alkynylor substituted alkynyl group. In certain embodiments, L includes analkoxy or substituted alkoxy group. In certain embodiments, L includesan amino or substituted amino group. In certain embodiments, L includesa carboxyl or carboxyl ester group. In certain embodiments, L includesan acyl amino group. In certain embodiments, L includes an alkylamide orsubstituted alkylamide group. In certain embodiments, L includes an arylor substituted aryl group. In certain embodiments, L includes aheteroaryl or substituted heteroaryl group. In certain embodiments, Lincludes a cycloalkyl or substituted cycloalkyl group. In certainembodiments, L includes a heterocyclyl or substituted heterocyclylgroup.

In certain embodiments, L includes a polymer. For example, the polymermay include a polyalkylene glycol and derivatives thereof, includingpolyethylene glycol, methoxypolyethylene glycol, polyethylene glycolhomopolymers, polypropylene glycol homopolymers, copolymers of ethyleneglycol with propylene glycol (e.g., where the homopolymers andcopolymers are unsubstituted or substituted at one end with an alkylgroup), polyvinyl alcohol, polyvinyl ethyl ethers, polyvinylpyrrolidone,combinations thereof, and the like. In certain embodiments, the polymeris a polyalkylene glycol. In certain embodiments, the polymer is apolyethylene glycol.

In some embodiments, L is a linker comprising-(L1)_(a)-(L²)_(b)-(L³)_(c)-(L⁴)_(d)-(L⁵)_(e)-, wherein L¹, L² , L³, L⁴and L⁵ are each a linker unit, and a, b, c, d and e are eachindependently 0 or 1, wherein the sum of a, b, c, d and e is 1 to 5.Other linkers are also possible, as shown in the conjugates andcompounds described in more detail herein.

In certain embodiments, W¹ is selected from a chemcial entity and apolypeptide. In certain embodiments, W¹ is a chemical entity (e.g., adrug or a detectable label). In certain embodiments, W¹ is a drug. Incertain embodiments, W¹ is a detectable label. In certain embodiments,W¹ is a polypeptide.

In certain instances, the hydrazinyl-pyrrolo compound is a compound offormula (VI):

wherein

Q³, Q⁴, X¹, X², X³, X⁴, L, W¹, Y¹, Y², and Y³ are as defined in formula(V).

As described above, in formula (V), in some instances, R¹⁶ is -L-W¹,which results in a compound of formula (VI) as shown above.

In certain embodiments, the substituents for formula (VI) are the sameas for formula (V) described above. For example, in certain embodiments,the substituents for formula (VI), e.g., Q³, Q⁴, X¹, X², X³, X⁴, L, W¹,Y¹, Y², and Y³ are as defined in formula (V).

In some embodiments, the compound is a compound of formula (VIIa) or(VIIb):

wherein:

X¹ and X³ are each independently O, S or NR¹²;

X⁴ is C;

in formula (VIIa), one of Q³ and Q⁴ is —(CH₂)_(m)NR³NHR² and the otheris Y⁴;

in formula (VIIb), one of Q³ and R¹², if present, is —(CH₂)_(m)NR³NHR²and the other is Y³;

Y¹, Y², Y³ and Y⁴, if present, are each independently selected fromhydrogen, halogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, amino acyl, alkylamide, substituted alkylamide, sulfonyl,thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, wherein Y¹ and Y² areoptionally cyclically linked, and wherein when Q⁴ is Y⁴, then Y³ and Y⁴are optionally cyclically linked; and n, R² and R³ are as defined informula (V).

In certain embodiments, the substituents for formulae (VIIa) and (VIIb)are the same as for formula (V) described above.

For example, in certain embodiments of formula (VIIa), X⁴ is C, and oneof Q³ and R¹² is —(CH₂)_(m)NR³NHR² and the other is Y³. In certainembodiments of formula (VIIa), X⁴ is C, Q³ is —(CH₂)_(m)NR³NHR² and Q⁴is Y³. In certain embodiments of formula (VIIa), X⁴ is C, Q⁴ is—(CH₂)_(m)NR³NHR² and Q³ is Y³. In certain embodiments, m is 0 or 1. Incertain embodiments, m is 0. In certain embodiments, m is 1.

In certain embodiments of formula (VIIb), X³ is NR¹², and one of Q³ andR¹² is —(CH₂)_(m)NR³NHR² and the other is Y³. In certain embodiments offormula (VIIb), X³ is NR¹², Q³ is —(CH₂)_(m)NR³NHR² and R¹² is Y³. Incertain embodiments of formula (VIIb), X³ is NR¹², R¹² is—(CH₂)_(m)NR³NHR² and Q³ is Y³. In certain embodiments, m is 0 or 1. Incertain embodiments, m is 0. In certain embodiments, m is 1.

In certain embodiments, Y¹ and Y² are optionally cyclically linked, andwherein when Q⁴ is Y⁴, then Y³ and Y⁴ are optionally cyclically linkedto form a fused benzo ring. In certain embodiments of formula (VIIb), Y¹and Y² are cyclically linked to form a fused benzo ring. In certainembodiments of formula (VIIa), Q⁴ is Y⁴ and Y³ and Y⁴ are cyclicallylinked to form a fused benzo ring.

In some embodiments, in the compounds, Q² is —(CH₂)_(m)NR³NHR² and Q³ isY⁴. In some embodiments, in the compounds, Q³ is —(CH₂)_(m)NR³NHR² andQ² is Y⁴. In certain embodiments, m is 1.

In some embodiments, R² and R³ are each independently selected fromalkyl and substituted alkyl. In certain embodiments, R² and R³ are eachmethyl.

In some embodiments, X¹, X², X³ and X⁴ are each C.

In some embodiments, Y¹, Y², Y³ and Y⁴ are each H.

In some embodiments, X¹ and X³ are each S and X⁴ is C. In someembodiments, X¹ and X³ are each O and X⁴ is C. In some embodiments offormula (VIIa), X¹ is S and X⁴ is C.

In some embodiments of formula (VIIa), X¹ is O and X⁴ is C. In someembodiments of formula (VIIb), X³ is S. In some embodiments of formula(VIIb), X³ is O.

In some embodiments, X¹ and X³ are each NR¹² and X⁴ is C. In someembodiments of formula (VIIa), X¹ is NR¹² and X⁴ is C. In someembodiments of formula (VIIa), X¹ is NH and X⁴ is C. In some embodimentsof formula (VIIb), X³ is NR¹². In some embodiments of formula (VIIb), X³is NH.

In some embodiments, W¹ is a chemical entity. In some embodiments, W¹ isa drug or a detectable label. In certain embodiments, the detectablelabel comprises a fluorophore. In some embodiments, W² is thepolypeptide.

In some embodiments, the compound is a compound of formula (VIII):

wherein

m is 0 or 1;

R² and R³ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, orR² and R³ are optionally cyclically linked to form a 5 or 6-memberedheterocyclyl;

X¹, X², X³ and X⁴ are each independently selected from C, N, O and S;

Y¹, Y², Y³ and Y⁴ are each independently selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl, wherein Y¹ and Y², Y² and Y³, or Y³ and Y⁴ areoptionally cyclically linked;

L is an optional linker (e.g., a linker as described herein); and

W¹ is selected from a chemical entity and a polypeptide.

In certain embodiments, the substituents for formula (VIII) are the sameas for formula (V) described above.

In some embodiments, the compound is a compound of formula (VIIIa):

In certain embodiments, the substituents in formula (VIIIa) are asdescribed above for formula (VIII).

Embodiments of the compound include a compound of formula (VIIIb):

where W¹ and L are as as described above for formula (VIII).

In some embodiments, the compound is a compound of formula (IX):

wherein

m is 0 or 1;

R² and R³ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, orR² and R³ are optionally cyclically linked to form a 5 or 6-memberedheterocyclyl;

X¹, X², X³ and X⁴ are each independently selected from C, N, O and S;

Y¹, Y², Y³ and Y⁴ are each independently selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl, wherein Y¹ and Y² or Y² and Y³ are optionallycyclically linked;

L is an optional linker (e.g., a linker as described herein); and

W¹ is selected from a chemical entity and a polypeptide.

In certain embodiments, the substituents in formula (IX) are asdescribed above for formula (V). For example, in certain embodiments, mis 0 or 1. In certain embodiments, m is 0. In certain embodiments, m is1.

In some embodiments, the compound is a compound of formula (IXa):

In certain embodiments, the substituents in formula (IXa) are asdescribed above for formula (IX).

Embodiments of the compound include a compound of formula (IXb):

In certain embodiments, the substituents in formula (IXb) are asdescribed above for formula (IX).

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

In certain embodiments, the compound is of the following structure:

Any of the chemical entities, linkers and coupling moieties set forth inthe structures above may be adapted for use in the subject compounds andconjugates.

Additional disclosure related to hydrazinyl-indole compounds and methodsfor producing a conjugate is found in U.S. application Ser. No.13/794,159, filed Mar. 11, 2013, the disclosure of which is incorporatedherein by reference.

Target Polypeptides

Any of a wide variety of polypeptides can be modified to be conjugatedto a second moiety as described above. Polypeptides suitable formodification include both proteins having a naturally-occurring aminoacid sequence, fragments of naturally-occurring polypeptides, andnon-naturally occurring polypeptides and fragments thereof.

The following are examples of classes and types of polypeptides whichare of interest for modification using the compounds and methodsdescribed herein to produce the polypeptide conjugates described herein.

Therapeutic Polypeptides

In certain embodiments, the methods of producing a conjugate are appliedto modification of polypeptides that may provide for a therapeuticbenefit, such as those polypeptides for which attachment to a moiety canprovide for one or more of, for example, an increase in serum half-life,a decrease in an adverse immune response, additional or alternatebiological activity or functionality, and the like, or other benefit orreduction of an adverse side effect. Where the therapeutic polypeptideis an antigen for a vaccine, modification can provide for an enhancedimmunogenicity of the polypeptide.

Examples of classes of therapeutic proteins include those that arecytokines, chemokines, growth factors, hormones, antibodies, andantigens. Further examples include, but are not limited to, thefollowing: erythropoietin (EPO, e.g., native EPO or synthetic EPO (see,e.g., US 2003/0191291), such as, but not limited to, e.g., PROCRIT®,EPREX®, or EPOGEN® (epoetin-a), ARANESP® (darbepoietin-a), NEORECORMON®,EPOGIN® (epoetin-β), and the like); a growth hormone (e.g., asomatotropin, e.g., GENOTROPIN®, NUTROPIN®, NORDITROPIN®, SAIZEN®,SEROSTIM®, HUMATROPE®, etc.); human growth hormone (hGH); bovine growthhormone (bGH); follicle stimulating hormone (FSH); interferon (e.g.,IFN-γ, IFN-α, IFN-β, IFN-ω; IFN-τ, consensus interferon, and the like);insulin (e.g., Novolin, Humulin, Humalog, Lantus, Ultralente, etc.),insulin-like growth factor (e.g., IGF-I, IGF-II); blood factors (e.g.,Factor X, tissue plasminogen activator (TPA), and the like, such as, butnot limited to, e.g., ACTIVASE® (alteplase) tissue plasminogenactivator, NOVOSEVEN® (recombinant human factor VIIa), Factor VIIa,Factor VIII (e.g., KOGENATEC), Factor IX, β-globin, hemoglobin, and thelike); colony stimulating factors (e.g., granulocyte-CSF (G-CSF, e.g.,NEUPOGEN® (filgrastim)), macrophage-CSF (M-CSF),granulocyte-macrophage-CSF (GM-CSF), Neulasta (pegfilgrastim),granulocyte-monocyte colony stimulating factor, megakaryocyte colonystimulating factor, and the like), transforming growth factors (e.g.,TGF-beta, TGF-alpha); interleukins (e.g., IL-1, IL-2 (e.g., Proleukin®),IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, and the like); a growthfactor (e.g., epidermal growth factor (EGF), platelet-derived growthfactor (PDGF, e.g., REGRANEX® (beclapermin)), fibroblast growth factors(FGFs, e.g., aFGF, bFGF, such as FIBLAST® (trafermin)), glial cellline-derived growth factor (GDNF), nerve growth factor (NGF), stem cellfactor (e.g., STEMGEN® (ancestim)), keratinocyte growth factor, ahepatocyte growth factor, and the like); a soluble receptor (e.g., aTNF-α-binding soluble receptor such as ENBREL® (etanercept), a solubleVEGF receptor, a soluble interleukin receptor, a soluble y/6 T cellreceptor, and the like); an enzyme (e.g., α-glucosidase, CERAZYME®(imiglucarase, 3-glucocerebrosidase, CEREDASE® (alglucerase); an enzymeactivator (e.g., tissue plasminogen activator); a chemokine (e.g.,IP-10, Mig, Groα/IL-8, regulated and normal T cell expressed andsecreted (RANTES), MIP-1α, MIP-10, MCP-1, PF-4, and the like); anangiogenic agent (e.g., vascular endothelial growth factor (VEGF); ananti-angiogenic agent (e.g., a soluble VEGF receptor); a proteinvaccine; a neuroactive peptide such as bradykinin, cholecystokinin,gastin, secretin, oxytocin, gonadotropin-releasing hormone,beta-endorphin, enkephalin, substance P, somatostatin, galanin, growthhormone-releasing hormone, bombesin, warfarin, dynorphin, neurotensin,motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin,insulin, glucagon, vasopres sin, angiotensin II, thyrotropin-releasinghormone, vasoactive intestinal peptide, a sleep peptide, etc.; otherproteins such as a thrombolytic agent, an atrial natriuretic peptide,bone morphogenic protein, thrombopoietin, relaxin, glial fibrillaryacidic protein, follicle stimulating hormone, a human alpha-1antitrypsin, a leukemia inhibitory factor, a transforming growth factor,a tissue factor, an insulin-like growth factor, a luteinizing hormone, afollicle stimulating hormone, a macrophage activating factor, tumornecrosis factor, a neutrophil chemotactic factor, a nerve growth factor,a tissue inhibitor of metalloproteinases; a vasoactive intestinalpeptide, angiogenin, angiotropin, fibrin; hirudin; a leukemia inhibitoryfactor; an IL-1 receptor antagonist (e.g., Kineret® (anakinra)); and thelike. It will be readily appreciated that native forms of the abovetherapeutic proteins are also of interest as target polypeptides in thepresent disclosure.

Further examples include antibodies, e.g., polyclonal antibodies,monoclonal antibodies, humanized antibodies, antigen-binding fragments(e.g., F(ab)′, Fab, Fv), single chain antibodies, and the like (e.g.,RITUXAN® (rituximab); REMICADE® (infliximab); HERCEPTINT® (trastuzumab);HUMIRA™ (adalimumab); XOLAIR® (omalizumab); BEXXAR® (tositumomab);RAPTIVA™ (efalizumab); ERBITUX™ (cetuximab); and the like). In someinstances, antibodies include antibodies that specifically bind to atumor antigen, an immune cell antigen (e.g., CD4, CD8, and the like), anantigen of a microorganism, particularly a pathogenic microorganism(e.g., a bacterial, viral, fungal, or parasitic antigen), and the like.

In some instances, the methods, conjugates and compounds describedherein can be applied to provide for a moiety (e.g., a water-solublepolymer) at a native or engineered site of glycosylation, such as foundin hyperglycosylated forms of a therapeutic protein.

The biological activity of a modified target polypeptide can be assayedaccording to methods known in the art. Modified polypeptides that retainat least one desired pharmacologic activity of the corresponding parentprotein are of interest.

Immunogenic Compositions

The methods, conjugates and compounds disclosed herein also findapplication in production of components of immunogenic compositions(e.g., therapeutic vaccines). For example, the compounds can be used tofacilitate attachment of moieties that increase serum half-life of apolypeptide antigen, that increase immunogenicity of the polypeptide, orthat link a non-amino acid antigen to a polypeptide carrier. In thisregard, the compounds can be used to facilitate modification ofmicrobial antigens (e.g., a bacterial, viral, fungal, or parasiticantigen), tumor antigens, and other antigens which are of interest foradministration to a subject to elicit an immune response in the subject.Also of interest is modification of antigens that are useful ineliciting antibodies which can be useful as research tools.

Further examples of polypeptides of interest for modification using thecompounds disclosed herein include those that are of interest fordetection or functional monitoring in an assay (e.g., as a researchtool, in a drug screening assay, and the like). Examples of polypeptidesof this type include receptors (e.g., G-protein coupled receptors(GPCRs, including orphan GPCRs)), receptor ligands (includingnaturally-occurring and synthetic), protein channels (e.g., ion channels(e.g., potassium channels, calcium channels, sodium channels, and thelike), and other polypeptides. In some embodiments, modification of cellsurface-associated polypeptides, such as transmembrane polypeptides) isof interest, for example where such modification is accomplished whilethe polypeptide is present in a membrane. Methods for modification of apolypeptide under physiological conditions are described further below.

Methods of Polypeptide Production

In general, the polypeptides described herein may be expressed inprokaryotes or eukaryotes in accordance with conventional ways,depending upon the purpose for expression. Thus, the present inventionfurther provides a host cell, e.g., a genetically modified host cellthat comprises a nucleic acid encoding a polypeptide.

Host cells for production (including large scale production) of anunconjugated or modified polypeptide suitable to form a conjugate asdescribed herein can be selected from any of a variety of available hostcells. Examples of host cells include those of a prokaryotic oreukaryotic unicellular organism, such as bacteria (e.g., Escherichiacoli strains, Bacillus spp. (e.g., B. subtilis), and the like) yeast orfungi (e.g., S. cerevisiae, Pichia spp., and the like), and other suchhost cells can be used. Examples of host cells originally derived from ahigher organism such as insects, vertebrates, including mammals, (e.g.,CHO, HEK, and the like), may be used as the expression host cells.

Suitable mammalian cell lines include, but are not limited to, HeLacells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHOcells (e.g., ATCC Nos. CRL9618 and CRL9096), CHO DG44 cells (Urlaub(1983) Cell 33:405), CHO-Ki cells (ATCC CCL-61), 293 cells (e.g., ATCCNo. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658),Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No.CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse Lcells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No.CRL1573), HLHepG2 cells, and the like.

Specific expression systems of interest include bacterial, yeast, insectcell and mammalian cell derived expression systems.

The expressed polypeptide can be recovered by any appropriate meansknown in the art. Further, any convenient protein purificationprocedures may be employed, where suitable protein purificationmethodologies are described in Guide to Protein Purification, (Deuthsered.) (Academic Press, 1990). For example, a lysate may prepared from acell comprising the expression vector expressing the desiredpolypeptide, and purified using high performance liquid chromatography(HPLC), exclusion chromatography, gel electrophoresis, affinitychromatography, and the like.

Methods for Modification of a Polypeptide

In certain embodiments, the polypeptide may be conjugated to a moiety ofinterest without first modifying the polypeptide. For instance, thepolypeptide may include one or more reactive groups suitable forconjugation to the moiety of interest (e.g., a moiety comprising acoupling moiety, such as a pyrazolinone or pyrazolinone derivative asdescribed herein). In other embodiments, the polypeptide may be modifiedbefore conjugation to the moiety of interest. Modification of thepolypeptide may produce a modified polypeptide that contains one or morereactive groups suitable for conjugation to the moiety of interest.

In some cases, the polypeptide may be modified at one or more amino acidresidues to provide one or more reactive groups suitable for conjugationto the moiety of interest (e.g., a moiety comprising a coupling moiety,such as a pyrazalinone or pyrazalinone derivative as described herein).For example, carbonyls introduced into a polypeptide can be selectivelyreacted with a-nucleophiles, such as aminooxy- and hydrazide-bearingcompounds. Chemistries selective for carbonyl functional groups on aprotein with enhanced kinetics, site selectivity and conjugate stabilitymay result in improved bioconjugates and provide access to new productsand therapeutic targets as disclosed herein.

In certain embodiments, the polypeptide may be modified to include areactive aldehyde group (e.g., a reactive aldehyde). A reactive aldehydemay be included in an “aldehyde tag” or “ald-tag”, which is meant torefer to an amino acid sequence derived from a sulfatase motif that hasbeen converted by action of a formylglycine generating enzyme (FGE) tocontain a 2-formylglycine residue (referred to herein as “fGly”). ThefGly residue generated by an FGE is also referred to in the literatureas a “formylglycine”. Stated differently, the term “aldehyde tag” isused herein to refer to an amino acid sequence comprising a “converted”sulfatase motif (i.e., a sulfatase motif in which the cysteine or theserine residue has been converted to fGly by action of an FGE, e.g.,L(fGly)TPSR). A converted sulfatase motif may be derived from an aminoacid sequence comprising an “unconverted” sulfatase motif (i.e., asulfatase motif in which the cysteine or serine residues has not beenconverted to fGly by an FGE, but is capable of being converted, e.g., anunconverted sulfatase motif with the sequence: L(C/S)TPSR). By“conversion” as used in the context of action of a formylglycinegenerating enzyme (FGE) on a sulfatase motif refers to biochemicalmodification of a cysteine or serine residue in a sulfatase motif to aformylglycine (fGly) residue (e.g., Cys to fGly, or Ser to fGly).Additional aspects of aldehyde tags and uses thereof in site-specificprotein modification are described in U.S. Pat. No. 7,985,783 and U.S.Application Publication No. 2011/0117621, filed Mar. 20, 2009, thedisclosures of each of which are incorporated herein by reference.

Conversion of a polypeptide to include fGly can be accomplished bycell-based (in vivo) or cell-free methods (in vitro). Similarly,modification of a polypeptide to produce a polypeptide suitable forconjugation (e.g., modification to produce a polypeptide containing areactive group suitable for conjugation) can be accomplished bycell-based (in vivo) or cell-free methods (in vitro).

Alternatively, isolated, unmodified polypeptides can be isolatedfollowing recombinant production in a host cell lacking a suitableenzyme or by synthetic production. The isolated polypeptide may then becontacted with a suitable enzyme under conditions to provide for thedesired modification of the polypeptide to include fGly. The polypeptidecan be unfolded by methods known in the art (e.g., using heat,adjustment of pH, chaotropic agents, (e.g., urea, and the like), organicsolvents (e.g., hydrocarbons: octane, benzene, chloroform), etc.) andthe denatured protein contacted with a suitable enzyme. The modifiedpolypeptide can then be refolded under suitable conditions.

In some cases, the modified polypeptide containing the fGly residue maybe conjugated to the moiety of interest by reaction of the fGly with acompound as described herein (e.g., a compound containing a couplingmoiety, such as a pyrazalinone or pyrazalinone derivative as describedherein). For example, an fGly-containing polypeptide may be isolatedfrom a production source (e.g., recombinant host cell production,synthetic production), and contacted with a reactive partner-containingdrug or other moiety (e.g., detectable label) under conditions suitableto provide for conjugation of the drug or other moiety to thepolypeptide. For example, the reactive partner-containing drug or othermoiety may include a reactive moiety (e.g., a pyrazalinone orpyrazalinone derivative as described herein). Thepyrazalinone-containing drug or other moiety may be reacted with thepolypeptide to produce a polypeptide conjugate as described herein. Forexample, FIG. 1 shows schematic drawings of polypeptide conjugatesaccording to embodiments of the present disclosure. FIG. 1A shows areaction scheme for the production of a polypeptide conjugate thatincludes a cyclic coupling moiety. In FIG. 1A, a polypeptide thatincludes an fGly is reacted with a cyclic coupling moiety (e.g., apyrazalinone or pyrazalinone derivative) to produce a polypeptideconjugate that includes the cyclic coupling moiety. FIG. 1B shows areaction scheme for the production of a polypeptide conjugate thatincludes an acyclic coupling moiety. In FIG. 1B, a polypeptide thatincludes an fGly is reacted with an acyclic coupling moiety to produce apolypeptide conjugate that includes the acyclic coupling moiety.

In some cases, the modified polypeptide containing the fGly residue maybe conjugated to the moiety of interest by reaction of the fGly with acompound such as, but not limited to, one of the following compouds:

Polypeptide Conjugates

The polypeptides can be subjected to conjugation to provide forattachment of a wide variety of moieties. Examples of moieties ofinterest include, but are not limited to, a drug, a detectable label, asmall molecule, a water-soluble polymer, a peptide, and the like (alsoreferred to a “payload” or “cargo” herein). Thus, the present disclosureprovides a polypeptide conjugate as described above.

The moiety of interest is provided as a component of a reactive partnerfor reaction with a residue of a polypeptide. In certain embodiments,the methods of polypeptide conjugation are compatible with reactionconditions suitable for the polypeptide. For example, the reactionconditions may include a reaction mixture that includes water. In somecases, the reaction mixture may have a pH compatible with thepolypeptide, such as, but not limited to, a pH of 4 to 11, or a pH of 5to 10, or a pH of 6 to 9, or a pH of 6 to 8. In certain instances, thereaction mixture has a pH of 7. In some embodiments, the reactionconditions are performed at a temperature compatible with thepolypeptide. For example, the reaction conditions may be at atemperature of 20° C. to 45° C., such as 25° C. to 40° C., or 30° C. to40° C., or 35° C. to 40° C. In some cases, the reaction conditions areat room temperature (e.g., 25° C.). In some instances, the reactionconditions are at a temperature of 37° C.

Provided the present disclosure, the ordinarily skilled artisan canreadily adapt any of a variety of moieties to provide a reactive partnerfor conjugation to a polypeptide as contemplated herein. The ordinarilyskilled artisan will appreciate that factors such as pH and sterichindrance (i.e., the accessibility of the modified amino acid residue toreaction with a reactive partner of interest) are of importance.Modifying reaction conditions to provide for optimal conjugationconditions is well within the skill of the ordinary artisan, and isroutine in the art. Where conjugation is conducted with a polypeptidepresent in or on a living cell, the conditions are selected so as to bephysiologically compatible. For example, the pH can be droppedtemporarily for a time sufficient to allow for the reaction to occur butwithin a period tolerated by the cell (e.g., from about 30 min to 1hour). Physiological conditions for conducting modification ofpolypeptides on a cell surface can be similar to those used in aketone-azide reaction in modification of cells bearing cell-surfaceazides (see, e.g., U.S. Pat. No. 6,570,040).

In certain embodiments, the present disclosure provides a polypeptideconjugate, where the polypeptide is an antibody. As such, embodimentsinclude an antibody conjugated to a moiety of interest, where anantibody conjugated to a moiety of interest is referred to as an“antibody conjugate.” An Ig polypeptide generally includes at least anIg heavy chain constant region or an Ig light chain constant region, andcan further include an Ig variable region (e.g., a V_(L) region and/or aV_(H) region). Ig heavy chain constant regions include Ig constantregions of any heavy chain isotype, non-naturally occurring Ig heavychain constant regions (including consensus Ig heavy chain constantregions). An Ig constant region can be modified to be conjugated to amoiety of interest, where the moiety of interest is present in oradjacent a solvent-accessible loop region of the Ig constant region.

In some cases, an antibody conjugate of the present disclosure caninclude: 1) Ig heavy chain constant region conjugated to one or moremoieties of interest, and an Ig light chain constant region conjugatedto one or more moieties of interest; 2) an Ig heavy chain constantregion conjugated to one or more moieties of interest, and an Ig lightchain constant region that is not conjugated to a moiety of interest; or3) an Ig heavy chain constant region that is not conjugated to a moietyof interest, and an Ig light chain constant region conjugated to one ormore moieties of interest. A subject antibody conjugate can also includevariable VH and/or VL domains. As described above, the one or moremoieties of interest may be conjugated to the Ig heavy chain constantregion or the Ig light chain constant region at a single amino acidresidue (e.g., one or two moieties of interest conjugated to a singleamino acid residue), or conjugated to the Ig heavy chain constant regionand/or the Ig light chain constant region at two or more different aminoacid residues.

An antibody conjugate of the present disclosure can include, as theconjugated moiety, any of a variety of compounds, as described herein,e.g., a drug (e.g., a peptide drug, a small molecule drug, and thelike), a water-soluble polymer, a detectable label, a synthetic peptide,etc.

An antibody conjugate can have any of a variety of antigen-bindingspecificities, as described above, including, e.g., an antigen presenton a cancer cell; an antigen present on an autoimmune cell; an antigenpresent on a pathogenic microorganism; an antigen present on avirus-infected cell (e.g., a human immunodeficiency virus-infectedcell), e.g., CD4 or gp120; an antigen present on a diseased cell; andthe like. For example, an antibody conjugate can bind an antigen, asnoted above, where the antigen is present on the surface of the cell. Anantibody conjugate of the present disclosure can bind antigen with asuitable binding affinity, e.g., from 5×10⁻⁶ M to 10⁻⁷ M, from 10⁻⁷ M to5×10⁻⁷ M, from 5×10⁻⁷ M to 10⁻⁸ M, from 10⁻⁸ M to 5×10⁻⁸ M, from 5×10⁻⁸M to 10⁻⁹ M, or a binding affinity greater than 10⁻⁹ M.

As non-limiting examples, a subject antibody conjugate can bind anantigen present on a cancer cell (e.g., a tumor-specific antigen; anantigen that is over-expressed on a cancer cell; etc.), and theconjugated moiety can be a cytotoxic compound (e.g., a cytotoxic smallmolecule, a cytotoxic synthetic peptide, etc.). For example, a subjectantibody conjugate can be specific for an antigen on a cancer cell,where the conjugated moiety is a cytotoxic compound (e.g., a cytotoxicsmall molecule, a cytotoxic synthetic peptide, etc.).

As further non-limiting examples, a subject antibody conjugate can bindan antigen present on a cell infected with a virus (e.g., where theantigen is encoded by the virus; where the antigen is expressed on acell type that is infected by a virus; etc.), and the conjugated moietycan be a viral fusion inhibitor. For example, a subject antibodyconjugate can bind an antigen present on a cell infected with a virus,and the conjugated moiety can be a viral fusion inhibitor.

Embodiments of the present disclosure also include polypeptideconjugates where the polypeptide is a carrier protein. For example,carrier proteins can be covalently and site-specifically bound to drugto provide a drug-containing scaffold. A carrier protein can besite-specifically conjugated to a covalently bound molecule of interest,such as a drug (e.g., a peptide, a small molecule drug, and the like),detectable label, etc. In certain embodiments, drug-scaffold conjugatescan provide for enhanced serum half-life of the drug.

In general a “carrier protein” is a protein that is biologically inert,is susceptible to modification as disclosed herein, and which canprovide for solvent-accessible presentation of the moiety of interestconjugated to the carrier protein through a modified amino acid residuein the carrier protein (e.g., through an oxime or hydrazone bond withinthe converted sulfatase motif of an aldehyde tagged carrier protein) ina physiological environment. “Biologically inert” is meant to indicatethe carrier protein exhibits clinically insignificant or no detectablebiological activity when administered to the appropriate subject, suchas when administered to a human subject. Thus, carrier proteins arebiologically inert in that they, for example, are of low immunogenicity,do not exhibit significant or detectable targeting properties (e.g., donot exhibit significant or detectable activity in binding to a specificreceptor), and exhibit little or no detectable biological activity thatmay interfere with activity of the moiety (e.g., drug or detectablelabel) conjugated to the aldehyde-tagged carrier protein. By “lowimmunogenicity” is meant that the carrier protein elicits little or nodetectable immune response upon administration to a subject, such as amammalian subject, e.g., a human subject. Carrier proteins can beprovided in monomeric or multimeric (e.g., dimeric) forms.

Carrier proteins having a three-dimensional structure when folded thatprovides for multiple different solvent-accessible sites that areamenable to modification (and thus conjugation to a moiety of interest)are of interest. In general, carrier proteins of interest are those thatare of a size and three-dimensional folded structure so as to providefor presentation of the conjugated moiety of interest on solventaccessible surfaces in a manner that is sufficiently spatially separatedso as to provide for activity and bioavailability of the conjugatedmoiety or moieties of interest. The carrier protein may be selectedaccording to a variety of factors including, but not limited to, themoiety (e.g., drug or detectable label) to be conjugated to the carrierprotein.

Accordingly, any of a wide variety of polypeptides can be suitable foruse as carrier proteins for use in the carrier protein conjugates of thepresent disclosure. Such carrier proteins can include those having anaturally-occurring amino acid sequence, fragments ofnaturally-occurring polypeptides, and non-naturally occurringpolypeptides and fragments thereof.

Examples of carrier proteins include, but are not limited to, albuminand fragments thereof (e.g., human serum albumin, bovine serum albumin,and the like), transferrin and fragments thereof (e.g. humantransferrin), and Fc fragments having reduced binding to a mammalian Fcreceptor, particularly a human Fc receptor (e.g., a modified Fc fragmentof an antibody (e.g., IgG), such as a mammalian antibody, e.g., a humanantibody). Examples of modified Fc fragments having reduced Fc receptorbinding are exemplified by the Fc fragments of Herceptin (trastuzumab)and Rituxan (Rituximab), which contain point mutations that provide forreduced Fc receptor binding (see, e.g., Clynes et al., Nature Medicine(2000), 6, 443-446). Alternatively or in addition, the isotype of the Fcfragment can be selected according to a desired level of Fc receptorbinding (e.g., use of an Fc fragment of an IgG4 isotype human heavychain constant region rather than from IgG1 or IgG3. (see, e.g., FridmanFASEB J 1991 September; 5 (12): 2684-90). In general, carrier proteinscan be at least about 4 kDa (e.g., about 50 amino acid residues inlength), usually at least about 25 kDa, and can be larger in size (e.g.,transferrin has a molecular weight of 90kDa while Fc fragments can havemolecular weights of 30kDa to 50kDa).

The conjugates described herein can be used for a variety ofapplications including, but not limited to, visualization usingfluorescence or epitope labeling (e.g., electron microscopy using goldparticles equipped with reactive groups for conjugation to the compoundsand conjugates described herein); protein immobilization (e.g., proteinmicroarray production); protein dynamics and localization studies andapplications; and conjugation of proteins with a moiety of interest(e.g., moieties that improve a parent protein's half-life (e.g.,poly(ethylene glycol)), targeting moieties (e.g., to enhance delivery toa site of action), and biologically active moieties (e.g., a therapeuticmoiety).

The polypeptide conjugate may include a polypeptide conjugated to amoiety or moieties that provide for one or more of a wide variety offunctions or features. In general, examples of moieties include, but arenot limited to, the following: detectable labels (e.g., fluorescentlabels); light-activated dynamic moieties (e.g., azobenzene mediatedpore closing, azobenzene mediated structural changes, photodecagingrecognition motifs); water soluble polymers (e.g., PEGylation);purification tags (e.g., to facilitate isolation by affinitychromatography (e.g., attachment of a FLAG epitope); membranelocalization domains (e.g., lipids or glycophosphatidylinositol(GPI)-type anchors); immobilization tags (e.g., to facilitate attachmentof the polypeptide to a surface, including selective attachment); drugs(e.g., to facilitate drug targeting, e.g., through attachment of thedrug to an antibody); targeted delivery moieties, (e.g., ligands forbinding to a target receptor (e.g., to facilitate viral attachment,attachment of a targeting protein present on a liposome, etc.)), and thelike.

Specific, non-limiting examples are provided below.

Drugs for Conjugation to a Polypeptide

Any of a number of drugs are suitable for use, or can be modified to berendered suitable for use, as a reactive partner to conjugate to apolypeptide. Examples of drugs include small molecule drugs and peptidedrugs. Thus, the present disclosure provides drug-polypeptideconjugates.

“Small molecule drug” as used herein refers to a compound, e.g., anorganic compound, which exhibits a pharmaceutical activity of interestand which is generally of a molecular weight of 800 Da or less, or 2000Da or less, but can encompass molecules of up to 5kDa and can be aslarge as 10 kDa. A small inorganic molecule refers to a moleculecontaining no carbon atoms, while a small organic molecule refers to acompound containing at least one carbon atom.

“Peptide drug” as used herein refers to amino-acid containing polymericcompounds, and is meant to encompass naturally-occurring andnon-naturally-occurring peptides, oligopeptides, cyclic peptides,polypeptides, and proteins, as well as peptide mimetics. The peptidedrugs may be obtained by chemical synthesis or be produced from agenetically encoded source (e.g., recombinant source). Peptide drugs canrange in molecular weight, and can be from 200 Da to 10 kDa or greaterin molecular weight.

In some cases, the drug is a cancer chemotherapeutic agent. For example,where the polypeptide is an antibody (or fragment thereof) that hasspecificity for a tumor cell, the antibody can be modified as describedherein to include a modified amino acid, which can be subsequentlyconjugated to a cancer chemotherapeutic agent. Cancer chemotherapeuticagents include non-peptidic (i.e., non-proteinaceous) compounds thatreduce proliferation of cancer cells, and encompass cytotoxic agents andcytostatic agents. Non-limiting examples of chemotherapeutic agentsinclude alkylating agents, nitrosoureas, antimetabolites, antitumorantibiotics, plant (vinca) alkaloids, and steroid hormones. Peptidiccompounds can also be used.

Suitable cancer chemotherapeutic agents include dolastatin and activeanalogs and derivatives thereof; and auristatin and active analogs andderivatives thereof (e.g., Monomethyl auristatin D (MMAD), monomethylauristatin E (MMAE), monomethyl auristatin F (MMAF), and the like). See,e.g., WO 96/33212, WO 96/14856, and U.S. Pat. No. 6,323,315. Forexample, dolastatin 10 or auristatin PE can be included in anantibody-drug conjugate of the present disclosure. Suitable cancerchemotherapeutic agents also include maytansinoids and active analogsand derivatives thereof (see, e.g., EP 1391213; and Liu et al (1996)Proc. Natl. Acad. Sci. USA 93:8618-8623); duocarmycins and activeanalogs and derivatives thereof (e.g., including the syntheticanalogues, KW-2189 and CB 1-TM1); and benzodiazepines and active analogsand derivatives thereof (e.g., pyrrolobenzodiazepine (PBD).

Agents that act to reduce cellular proliferation are known in the artand widely used. Such agents include alkylating agents, such as nitrogenmustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, andtriazenes, including, but not limited to, mechlorethamine,cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine(BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin,chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil,pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan,dacarbazine, and temozolomide.

Antimetabolite agents include folic acid analogs, pyrimidine analogs,purine analogs, and adenosine deaminase inhibitors, including, but notlimited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil(5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP),pentostatin, 5-fluorouracil (5-FU), methotrexate,10-propargyl-5,8-dideazafolate (PDDF, CB3717),5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabinephosphate, pentostatine, and gemcitabine.

Suitable natural products and their derivatives, (e.g., vinca alkaloids,antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins),include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel(Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine;brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine,vindesine, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc.;antibiotics, e.g. anthracycline, daunorubicin hydrochloride (daunomycin,rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin andmorpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g.dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinoneglycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g.mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; macrocyclicimmunosuppressants, e.g. cyclosporine, FK-506 (tacrolimus, prograf),rapamycin, etc.; and the like.

Other anti-proliferative cytotoxic agents are navelbene, CPT-11,anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide,ifosamide, and droloxafine.

Microtubule affecting agents that have antiproliferative activity arealso suitable for use and include, but are not limited to,allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine(NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel(Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC361792), trityl cysterin, vinblastine sulfate, vincristine sulfate,natural and synthetic epothilones including but not limited to,eopthilone A, epothilone B, discodermolide; estramustine, nocodazole,and the like.

Hormone modulators and steroids (including synthetic analogs) that aresuitable for use include, but are not limited to, adrenocorticosteroids,e.g. prednisone, dexamethasone, etc.; estrogens and pregestins, e.g.hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrolacetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocorticalsuppressants, e.g. aminoglutethimide; 17α-ethinylestradiol;diethylstilbestrol, testosterone, fluoxymesterone, dromostanolonepropionate, testolactone, methylprednisolone, methyl-testosterone,prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone,aminoglutethimide, estramustine, medroxyprogesterone acetate,leuprolide, Flutamide (Drogenil), Toremifene (Fareston), and Zoladex®.Estrogens stimulate proliferation and differentiation; thereforecompounds that bind to the estrogen receptor are used to block thisactivity. Corticosteroids may inhibit T cell proliferation.

Other suitable chemotherapeutic agents include metal complexes, e.g.cisplatin (cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; andhydrazines, e.g. N-methylhydrazine; epidophyllotoxin; a topoisomeraseinhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; etc. Otheranti-proliferative agents of interest include immunosuppressants, e.g.mycophenolic acid, thalidomide, desoxyspergualin, azasporine,leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline);etc.

Taxanes are suitable for use. “Taxanes” include paclitaxel, as well asany active taxane derivative or pro-drug. “Paclitaxel” (which should beunderstood herein to include analogues, formulations, and derivativessuch as, for example, docetaxel, TAXOL™, TAXOTERE™ (a formulation ofdocetaxel), 10-desacetyl analogs of paclitaxel and3′N-desbenzoyl-3′N-t-butoxycarbonyl analogs of paclitaxel) may bereadily prepared utilizing techniques known to those skilled in the art(see also WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO93/23555, WO 93/10076; U.S. Pat. Nos. 5,294,637; 5,283,253; 5,279,949;5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267), or obtainedfrom a variety of commercial sources, including for example, SigmaChemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; or T-1912from Taxus yannanensis).

Paclitaxel should be understood to refer to not only the commonchemically available form of paclitaxel, but analogs and derivatives(e.g., Taxotere™ docetaxel, as noted above) and paclitaxel conjugates(e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

Also included within the term “taxane” are a variety of knownderivatives, including both hydrophilic derivatives, and hydrophobicderivatives. Taxane derivatives include, but not limited to, galactoseand mannose derivatives described in International Patent ApplicationNo. WO 99/18113; piperazino and other derivatives described in WO99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, andU.S. Pat. No. 5,869,680; 6-thio derivatives described in WO 98/28288;sulfenamide derivatives described in U.S. Pat. No. 5,821,263; and taxolderivative described in U.S. Pat. No. 5,415,869. It further includesprodrugs of paclitaxel including, but not limited to, those described inWO 98/58927; WO 98/13059; and U.S. Pat. No. 5,824,701.

Biological response modifiers suitable for use include, but are notlimited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2)inhibitors of serine/threonine kinase activity; (3) tumor-associatedantigen antagonists, such as antibodies that bind specifically to atumor antigen; (4) apoptosis receptor agonists; (5) interleukin-2; (6)IFN-α; (7) IFN-γ; (8) colony-stimulating factors; and (9) inhibitors ofangiogenesis.

Methods for Modification of Drugs to Contain a Reactive Partner

Drugs to be conjugated to a polypeptide may be modified to incorporate areactive partner for reaction with the polypeptide. Where the drug is apeptide drug, the reactive moiety (e.g., aminooxy or hydrazide can bepositioned at an N-terminal region, the N-terminus, a C-terminal region,the C-terminus, or at a position internal to the peptide. For example,an example of a method involves synthesizing a peptide drug having anaminooxy group. In this example, the peptide is synthesized from aBoc-protected precursor. An amino group of a peptide can react with acompound comprising a carboxylic acid group and oxy-N-Boc group. As anexample, the amino group of the peptide reacts with3-(2,5-dioxopyrrolidin-1-yloxy)propanoic acid. Other variations on thecompound comprising a carboxylic acid group and oxy-N-protecting groupcan include different number of carbons in the alkylene linker andsubstituents on the alkylene linker. The reaction between the aminogroup of the peptide and the compound comprising a carboxylic acid groupand oxy-N-protecting group occurs through standard peptide couplingchemistry. Examples of peptide coupling reagents that can be usedinclude, but not limited to, DCC (dicyclohexylcarbodiimide), DIC(diisopropylcarbodiimide), di-p-toluoylcarbodiimide, BDP(1-benzotriazolediethylphosphate-1-cyclohexyl-3-(2-morpholinylethyl)carbodiimide), EDC(1-(3-dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuricfluoride, cyanuric chloride, TFFH (tetramethyl fluoroformamidiniumhexafluorophosphosphate), DPPA (diphenylphosphorazidate), BOP(benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate), HBTU(O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate),TBTU (O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumtetrafluoroborate), TSTU(O-(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate),HATU(N-[(dimethylamino)-1-H-1,2,3-triazolo[4,5,6]-pyridin-1-ylmethylene]-N-methylmethanaminiumhexafluorophosphate N-oxide), BOP-Cl(bis(2-oxo-3-oxazolidinyl)phosphinic chloride), PyB OP((l-H-1,2,3-benzotriazol-1-yloxy)-tris(pyrrolidino)phosphoniumtetrafluorophopsphate), BrOP (bromotris(dimethylamino)phosphoniumhexafluorophosphate), DEPBT(3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one) PyBrOP(bromotris(pyrrolidino)phosphonium hexafluorophosphate). As anon-limiting example, HOBt and DIC can be used as peptide couplingreagents.

Deprotection to expose the amino-oxy functionality is performed on thepeptide comprising an N-protecting group. Deprotection of theN-oxysuccinimide group, for example, occurs according to standarddeprotection conditions for a cyclic amide group. Deprotectingconditions can be found in Greene and Wuts, Protective Groups in OrganicChemistry, 3rd Ed., 1999, John Wiley & Sons, NY and Harrison et al.Certain deprotection conditions include a hydrazine reagent, aminoreagent, or sodium borohydride. Deprotection of a Boc protecting groupcan occur with TFA. Other reagents for deprotection include, but are notlimited to, hydrazine, methylhydrazine, phenylhydrazine, sodiumborohydride, and methylamine. The product and intermediates can bepurified by conventional means, such as HPLC purification.

The ordinarily skilled artisan will appreciate that factors such as pHand steric hindrance (i.e., the accessibility of the amino acid residueto reaction with a reactive partner of interest) are of importance,Modifying reaction conditions to provide for optimal conjugationconditions is well within the skill of the ordinary artisan, and isroutine in the art. Where conjugation is conducted with a polypeptidepresent in or on a living cell, the conditions are selected so as to bephysiologically compatible. For example, the pH can be droppedtemporarily for a time sufficient to allow for the reaction to occur butwithin a period tolerated by the cell (e.g., from about 30 min to 1hour). Physiological conditions for conducting modification ofpolypeptides on a cell surface can be similar to those used in aketone-azide reaction in modification of cells bearing cell-surfaceazides (see, e.g., U.S. Pat. No. 6,570,040).

Small molecule compounds containing, or modified to contain, ana-nucleophilic group that serves as a reactive partner with a compoundor conjugate disclosed herein are also contemplated for use as drugs inthe polypeptide-drug conjugates of the present disclosure. Generalmethods are known in the art for chemical synthetic schemes andconditions useful for synthesizing a compound of interest (see, e.g.,Smith and March, March's Advanced Organic Chemistry: Reactions,Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; orVogel, A Textbook of Practical Organic Chemistry, Including QualitativeOrganic Analysis, Fourth Edition, New York: Longman, 1978).

Peptide Drugs

In some cases, a conjugate comprises a covalently linked peptide.Suitable peptides include, but are not limited to, cytotoxic peptides;angiogenic peptides; anti-angiogenic peptides; peptides that activate Bcells; peptides that activate T cells; anti-viral peptides; peptidesthat inhibit viral fusion; peptides that increase production of one ormore lymphocyte populations; anti-microbial peptides; growth factors;growth hormone-releasing factors; vasoactive peptides; anti-inflammatorypeptides; peptides that regulate glucose metabolism; an anti-thromboticpeptide; an anti-nociceptive peptide; a vasodilator peptide; a plateletaggregation inhibitor; an analgesic; and the like.

In some embodiments, the peptide can be chemically synthesized toinclude a group reactive with an amino acid residue or a modified aminoacid residue of the polypeptide. A suitable synthetic peptide has alength of from 5 amino acids to 100 amino acids, or longer than 100amino acids; e.g., a suitable peptide has a length of from 5 amino acids(aa) to 10 aa, from 10 aa to 15 aa, from 15 aa to 20 aa, from 20 aa to25 aa, from 25 aa to 30 aa, from 30 aa to 40 aa, from 40 aa to 50 aa,from 50 aa to 60 aa, from 60 aa to 70 aa, from 70 aa to 80 aa, from 80aa to 90 aa, or from 90 aa to 100 aa.

In certain embodiments, a peptide can be modified to contain ana-nucleophile-containing moiety (e.g., an aminooxy or hydrazide moiety),e.g., can be reacted with an fGly-containing polypeptide to yield aconjugate in which the polypeptide and peptide are linked by a hydrazoneor oxime bond, respectively. Examples of methods of synthesizing apeptide, such that the synthetic peptide comprising a reactive groupreactive with an amino acid residue or a modified amino acid residue ofthe polypeptide, are described above.

Suitable peptides include, but are not limited to, hLF-11 (an 11-aminoacid N-terminal fragment of lactoferrin), an anti-microbial peptide;granulysin, an anti-microbial peptide; Plectasin (NZ2114; SAR 215500),an anti-microbial peptide; viral fusion inhibitors such as Fuzeon(enfuvirtide), TRI-1249 (T-1249; see, e.g., Matos et al. (2010) PLoS One5:e9830), RI-2635 (T-2635; see, e.g., Eggink et al. (2009) J. Biol.Chem. 284:26941), T651, and TRI-1144; C5a receptor inhibitors such asPMX-53, JPE-1375, and JSM-7717; POT-4, a human complement factor C3inhibitor; Pancreate (an INGAP derivative sequence, a HIP-human proisletprotein); somatostatin; a somatostatin analog such as DEBIO 8609(Sanvar), octreotide, octreotide (C2L), octreotide QLT, octreotide LAR,Sandostatin LAR, SomaLAR, Somatuline (lanreotide), see, e.g., Deghenghiet al. (2001) Endocrine 14:29; TH9507 (Tesamorelin, a growthhormone-releasing factor); P0L7080 (a protegrin analog, ananti-microbial peptide); relaxin; a corticotropin releasing factoragonist such as urotensin, sauvagine, and the like; a heat shock proteinderivative such as DiaPep277; a human immunodeficiency virus entryinhibitor; a heat shock protein-20 mimic such as AZX100; a thrombinreceptor activating peptide such as TP508 (Chrysalin); a urocortin 2mimic (e.g., a CRF2 agonist) such as urocortin-2; an immune activatorsuch as Zadaxin (thymalfasin; thymosin-α1), see, e.g., Sjogren (2004) J.Gastroenterol. Hepatol. 19:S69; a hepatitis C virus (HCV) entryinhibitorE2 peptide such as HCV3; an atrial natriuretic peptide such asHANP (Sun 4936; carperitide); an annexin peptide; a defensin(anti-microbial peptide) such as hBD2-4; a defensin (anti-microbialpeptide) such as hBD-3; a defensin (anti-microbial peptide) such asPMX-30063; a histatin (anti-microbial peptide) such as histatin-3,histatin-5, histatin-6, and histatin-9; a histatin (anti-microbialpeptide) such as PAC-113; an indolicidin (anti-microbial peptide) suchas MX-594AN (Omniganin; CLS001); an indolicidin (anti-microbial peptide)such as Omnigard (MBI-226; CPI-226); an anti-microbial peptide such asan insect cecropin; an anti-microbial peptide such as a lactoferrin(talactoferrin); an LL-37/cathelicidin derivative (an anti-microbialpeptide) such as P60.4 (OP-145); a magainin (an anti-microbial peptide)such as Pexiganan (MSI-78; Suponex); a protegrin (an anti-microbialpeptide) such as IB-367 (Iseganan); an agan peptide; a beta-natriureticpeptide such as Natrecor, or Noratak (Nesiritide), or ularitide;bivalarudin (Angiomax), a thrombin inhibitor; a C peptide derivative; acalcitonin such as Miacalcin (Fortical); an enkephalin derivative; anerythropoiesis-stimulating peptide such as Hematide; a gap junctionmodulator such as Danegaptide (ZP1609); a gastrin-releasing peptide; aghrelin; a glucagon-like peptide; a glucagon-like peptide-2 analog suchas ZP1846 or ZP1848; a glucosaminyl muramyl dipeptide such as GMDP; aglycopeptide antibiotic such as Oritavancin; a teicoplanin derivativesuch as Dalbavancin; a gonadotropin releasing hormone (GnRH) such asZoladex (Lupon) or Triptorelin; a histone deacetylase (HDAC) inhibitordepsipeptide such as PM02734 (Irvalec); an integrin such aseptifibatide; an insulin analog such as Humulog; a kahalalidedepsipeptide such as PM02734; a kallikrein inhibitor such as Kalbitor(ecallantide); an antibiotic such as Telavancin; a lipopeptide such asCubicin or MX-2401; a lutenizing hormone releasing hormone (LHRH) suchas goserelin; an LHRH synthetic decapeptide agonist analog such asTreistar (triptorelin pamoate); an LHRH such as Eligard; an M2 proteinchannel peptide inhibitor; metreleptin; a melanocortin receptor agonistpeptide such as bremalanotide/PT-141; a melanocortin; a muramyltripeptide such as Mepact (mifamurtide); a myelin basic protein peptidesuch as MBP 8298 (dirucotide); an N-type voltage-gated calcium channelblocker such as Ziconotide (Prialt); a parathyroid hormone peptide; aparathyroid analog such as 768974; a peptide hormone analog such asUGP281; a prostaglandin F2-a receptor inhibitor such as PDC31; aprotease inhibitor such as PPL-100; surfaxin; a thromobspondin-1 (TSP-1)mimetic such as CVX-045 or ABT 510; a vasoactive intestinal peptide;vasopressin; a Y2R agonist peptide such as RG7089; obinepeptide; andTM30339.

Detectable Labels

The conjugates, compounds and methods of the present disclosure can beused to conjugate a detectable label to polypeptide. Examples ofdetectable labels include, but are not limited to, fluorescent molecules(e.g., autofluorescent molecules, molecules that fluoresce upon contactwith a reagent, etc.), radioactive labels (e.g., ¹¹¹In, ¹²⁵In, ¹³¹I,²¹²B, ⁹⁰Y, ¹⁸⁶Rh, and the like), biotin (e.g., to be detected throughreaction of biotin and avidin), fluorescent tags, imaging reagents, andthe like. Detectable labels also include peptides or polypeptides thatcan be detected by antibody binding, e.g., by binding of a detectablylabeled antibody or by detection of bound antibody through asandwich-type assay. Further examples of detectable labels include, butare not limited to, dye labels (e.g., chromophores, fluorophores, suchas, but not limited to, Alexa Fluor® fluorescent dyes (e.g., AlexaFluor® 350, 405, 430, 488, 532, 546, 555, 568, 594, 595, 610, 633, 635,647, 660, 680, 700, 750, 790, and the like), coumarins, rhodamines(5-carboxyrhodamine and sulfo derivates thereof, e.g.,5-carboxy-disulfo-rhodamine, carbopyranins and oxazines, such as ATTOdyes (e.g., ATTO 390, 425, 465, 488, 495, 520, 532, 550, 565, 590, 594,610, 611X, 620, 633, 635, 637, 647, 647N, 655, 665, 680, 700, 725 or740), biophysical probes (spin labels, nuclear magnetic resonance (NMR)probes), Frster Resonance Energy Transfer (FRET)-type labels (e.g., atleast one member of a FRET pair, including at least one member of afluorophore/quencher pair), Bioluminescence Resonance Energy Transfer(BRET)-type labels (e.g., at least one member of a BRET pair),immunodetectable tags (e.g., FLAG, His(6), and the like), localizationtags (e.g., to identify association of a tagged polypeptide at thetissue or molecular cell level (e.g., association with a tissue type, orparticular cell membrane), and the like.

Attachment of Moieties for Delivery to a Target Site

Embodiments of the present disclosure also include a polypeptideconjugated to one or more moieties, such as, but not limited to, a drug(e.g., a small molecule drug), toxin, or other molecule for delivery toa target site (e.g., a cell) and which can provide for a pharmacologicalactivity or can serve as a target for delivery of other molecules.

Also contemplated are conjugates that include one of a pair of bindingpartners (e.g., a ligand, a ligand-binding portion of a receptor, areceptor-binding portion of a ligand, etc.). For example, the conjugatecan include a polypeptide that serves as a viral receptor and, uponbinding with a viral envelope protein or viral capsid protein,facilitates attachment of virus to the cell surface on which themodified polypeptide is expressed. Alternatively, the conjugate mayinclude an antigen that is specifically bound by an antibody (e.g.,monoclonal antibody), to facilitate detection and/or separation of hostcells expressing the modified polypeptide.

Attachment of Target Molecules to a Support

The methods can provide for conjugation of a polypeptide to a moiety tofacilitate attachment of the polypeptide to a solid substrate (e.g., tofacilitate assays), or to a moiety to facilitate easy separation (e.g.,a hapten recognized by an antibody bound to a magnetic bead). In someembodiments, the methods are used to provide for attachment of a proteinto an array (e.g., chip) in a defined orientation. For example, apolypeptide modified at a selected site (e.g., at or near theN-terminus) can be generated, and the methods, conjugates and compoundsused to deliver a moiety to the modified polypeptide. The moiety canthen be used as the attachment site for affixing the polypeptide to asupport (e.g., solid or semi-solid support, such as a support suitablefor use as a microchip in high-throughput assays).

Water-Soluble Polymers

In some cases, a conjugate includes a covalently linked water-solublepolymer. A moiety of particular interest is a water-soluble polymer. A“water-soluble polymer” refers to a polymer that is soluble in water andis usually substantially non-immunogenic, and usually has an atomicmolecular weight greater than 1,000 Daltons. The methods, conjugates andcompounds described herein can be used to attach one or morewater-soluble polymers to a polypeptide. Attachment of a water-solublepolymer (e.g., PEG) to a polypeptide, such as a pharmaceutically active(e.g., therapeutic) polypeptide can be desirable as such modificationcan increase the therapeutic index by increasing serum half-life as aresult of increased proteolytic stability and/or decreased renalclearance. Additionally, attachment of one or more polymers (e.g.,PEGylation) can reduce immunogenicity of protein pharmaceuticals.

In some embodiments, the water-soluble polymer has an effectivehydrodynamic molecular weight of greater than 5,000 Da, greater than10,000 Da, greater than 20,000 to 500,000 Da, greater than 40,000 Da to300,000 Da, greater than 50,000 Da to 70,000 Da, such as greater than60,000 Da. In some embodiments, the water-soluble polymer has aneffective hydrodynamic molecular weight of from 10 kDa to 20 kDa, from20 kDa to 25 kDa, from 25 kDa to 30 kDa, from 30 kDa to 50 kDa, or from50 kDa to 100 kDa. By “effective hydrodynamic molecular weight” isintended the effective water-solvated size of a polymer chain asdetermined by aqueous-based size exclusion chromatography (SEC). Whenthe water-soluble polymer contains polymer chains having polyalkyleneoxide repeat units, such as ethylene oxide repeat units, each chain canhave an atomic molecular weight of 200 Da to 80,000 Da, or 1,500 Da to42,000 Da, including 2,000 to 20,000 Da. Unless referred tospecifically, molecular weight is intended to refer to atomic molecularweight. Linear, branched, and terminally charged water soluble polymers(e.g., PEG) may be used.

Polymers useful as moieties to be attached to a polypeptide can have awide range of molecular weights, and polymer subunits. These subunitsmay include a biological polymer, a synthetic polymer, or a combinationthereof. Examples of such water-soluble polymers include: dextran anddextran derivatives, including dextran sulfate, P-amino cross linkeddextrin, and carboxymethyl dextrin, cellulose and cellulose derivatives,including methylcellulose and carboxymethyl cellulose, starch anddextrines, and derivatives and hydroylactes of starch, polyalklyeneglycol and derivatives thereof, including polyethylene glycol,methoxypolyethylene glycol, polyethylene glycol homopolymers,polypropylene glycol homopolymers, copolymers of ethylene glycol withpropylene glycol, wherein said homopolymers and copolymers areunsubstituted or substituted at one end with an alkyl group, heparin andfragments of heparin, polyvinyl alcohol and polyvinyl ethyl ethers,polyvinylpyrrolidone, aspartamide, and polyoxyethylated polyols, withthe dextran and dextran derivatives, dextrine and dextrine derivatives.It will be appreciated that various derivatives of the specificallyrecited water-soluble polymers are also contemplated.

Water-soluble polymers such as those described above includepolyalkylene oxide based polymers, such as polyethylene glycol “PEG”(See. e.g., “Poly(ethylene glycol) Chemistry: Biotechnical andBiomedical Applications”, J. M. Harris, Ed., Plenum Press, New York,N.Y. (1992); and “Poly(ethylene glycol) Chemistry and BiologicalApplications”, J. M. Harris and S. Zalipsky, Eds., ACS (1997); andInternational Patent Applications: WO 90/13540, WO 92/00748, WO92/16555, WO 94/04193,WO 94/14758, WO 94/17039, WO 94/18247, WO94/28937, WO 95/11924, WO 96/00080, WO 96/23794, WO 98/07713, WO98/41562, WO 98/48837, WO 99/30727, WO 99/32134, WO 99/33483, WO99/53951, WO 01/26692, WO 95/13312, WO 96/21469, WO 97/03106, WO99/45964, and U.S. Pat. Nos. 4,179,337; 5,075,046; 5,089,261; 5,100,992;5,134,192; 5,166,309; 5,171,264; 5,213,891; 5,219,564; 5,275,838;5,281,698; 5,298,643; 5,312,808; 5,321,095; 5,324,844; 5,349,001;5,352,756; 5,405,877; 5,455,027; 5,446,090; 5,470,829; 5,478,805;5,567,422; 5,605,976; 5,612,460; 5,614,549; 5,618,528; 5,672,662;5,637,749; 5,643,575; 5,650,388; 5,681,567; 5,686,110; 5,730,990;5,739,208; 5,756,593; 5,808,096; 5,824,778; 5,824,784; 5,840,900;5,874,500; 5,880,131; 5,900,461; 5,902,588; 5,919,442; 5,919,455;5,932,462; 5,965,119; 5,965,566; 5,985,263; 5,990,237; 6,011,042;6,013,283; 6,077,939; 6,113,906; 6,127,355; 6,177,087; 6,180,095;6,194,580; 6,214,966).

Examples of polymers of interest include those containing a polyalkyleneoxide, polyamide alkylene oxide, or derivatives thereof, includingpolyalkylene oxide and polyamide alkylene oxide comprising an ethyleneoxide repeat unit of the formula —(CH₂—CH₂—O)—. Further examples ofpolymers of interest include a polyamide having a molecular weightgreater than 1,000 Daltons of the formula —[C(O)—X—C(O)—NH—Y—NH]n- or—[NH—Y—NH—C(O)—X—C(O)]_(n)—, where X and Y are divalent radicals thatmay be the same or different and may be branched or linear, and n is adiscrete integer from 2-100, such as from 2 to 50, and where either orboth of X and Y comprises a biocompatible, substantially non-antigenicwater-soluble repeat unit that may be linear or branched. Furtherexamples of water-soluble repeat units comprise an ethylene oxide of theformula —(CH₂—CH₂—O)— or —(O—CH₂—CH₂)—. The number of such water-solublerepeat units can vary significantly, with the number of such units beingfrom 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, for example from2 to 50. An example of an embodiment is one in which one or both of Xand Y is selected from: —((CH₂)_(n1)—(CH₂—CH₂—O)_(n2)—(CH₂)— or—((CH₂)_(n1)—(O—CH₂—CH₂),₁₂—(CH₂)_(n1)—), where n1 is 1 to 6, 1 to 5, 1to 4, or 1 to 3, and where n2 is 2 to 50, 2 to 25, 2 to 15, 2 to 10, 2to 8, or 2 to 5. A further example of an embodiment is one in which X is—(CH₂—CH₂)—, and where Y is —(CH₂—(CH₂—CH₂—O)₃—CH₂—CH₂—CH₂)— or—(CH₂—CH₂—CH₂—(O—CH₂-CH₂)₃—CH₂)—.

The polymer can include one or more spacers or linkers. Examples ofspacers or linkers include linear or branched moieties comprising one ormore repeat units employed in a water-soluble polymer, diamino and ordiacid units, natural or unnatural amino acids or derivatives thereof,as well as aliphatic moieties, including alkyl, aryl, heteroalkyl,heteroaryl, alkoxy, and the like, which can contain, for example, up to18 carbon atoms or even an additional polymer chain.

The polymer moiety, or one or more of the spacers or linkers of thepolymer moiety when present, may include polymer chains or units thatare biostable or biodegradable. For example, polymers with repeatlinkages have varying degrees of stability under physiologicalconditions depending on bond lability. Polymers with such bonds can becategorized by their relative rates of hydrolysis under physiologicalconditions based on known hydrolysis rates of low molecular weightanalogs, e.g., from less stable to more stable, e.g., polyurethanes(—NH—C(O)—O—)> polyorthoesters (—O—C((OR)(R′))—O—)> polyamides(—C(O)—NH—). Similarly, the linkage systems attaching a water-solublepolymer to a target molecule may be biostable or biodegradable, e.g.,from less stable to more stable: carbonate (—O—C(O)—O—)> ester(—C(O)—O—)> urethane (—NH—C(O)—O—)> orthoester (—O—C((OR)(R′))—O—)>amide (—C(O)—NH—). In general, it may be desirable to avoid use of asulfated polysaccharide, depending on the lability of the sulfate group.In addition, it may be less desirable to use polycarbonates andpolyesters. These bonds are provided by way of example, and are notintended to limit the types of bonds employable in the polymer chains orlinkage systems of the water-soluble polymers useful in the modifiedaldehyde tagged polypeptides disclosed herein.

Formulations

The conjugates (including antibody conjugates) of the present disclosurecan be formulated in a variety of different ways. In general, where theconjugate is a polypeptide-drug conjugate, the conjugate is formulatedin a manner compatible with the drug conjugated to the polypeptide, thecondition to be treated, and the route of administration to be used.

The conjugate (e.g., polypeptide-drug conjugate) can be provided in anysuitable form, e.g., in the form of a pharmaceutically acceptable salt,and can be formulated for any suitable route of administration, e.g.,oral, topical or parenteral administration. Where the conjugate isprovided as a liquid injectable (such as in those embodiments where theyare administered intravenously or directly into a tissue), the conjugatecan be provided as a ready-to-use dosage form, or as a reconstitutablestorage-stable powder or liquid composed of pharmaceutically acceptablecarriers and excipients.

Methods for formulating conjugates can be adapted from those availablein the art. For example, conjugates can be provided in a pharmaceuticalcomposition comprising a therapeutically effective amount of a conjugateand a pharmaceutically acceptable carrier (e.g., saline). Thepharmaceutical composition may optionally include other additives (e.g.,buffers, stabilizers, preservatives, and the like). In some embodiments,the formulations are suitable for administration to a mammal, such asthose that are suitable for administration to a human.

Methods of Treatment

The polypeptide-drug conjugates of the present disclosure find use intreatment of a condition or disease in a subject that is amenable totreatment by administration of the parent drug (i.e., the drug prior toconjugation to the polypeptide). By “treatment” is meant that at leastan amelioration of the symptoms associated with the condition afflictingthe host is achieved, where amelioration is used in a broad sense torefer to at least a reduction in the magnitude of a parameter, e.g.symptom, associated with the condition being treated. As such, treatmentalso includes situations where the pathological condition, or at leastsymptoms associated therewith, are completely inhibited, e.g., preventedfrom happening, or stopped, e.g. terminated, such that the host nolonger suffers from the condition, or at least the symptoms thatcharacterize the condition. Thus treatment includes: (i) prevention,that is, reducing the risk of development of clinical symptoms,including causing the clinical symptoms not to develop, e.g., preventingdisease progression to a harmful state; (ii) inhibition, that is,arresting the development or further development of clinical symptoms,e.g., mitigating or completely inhibiting an active disease; and/or(iii) relief, that is, causing the regression of clinical symptoms.

In the context of cancer, the term “treating” includes any or all of:reducing growth of a solid tumor, inhibiting replication of cancercells, reducing overall tumor burden, and ameliorating one or moresymptoms associated with a cancer.

The subject to be treated can be one that is in need of therapy, wherethe host to be treated is one amenable to treatment using the parentdrug. Accordingly, a variety of subjects may be amenable to treatmentusing the polypeptide-drug conjugates disclosed herein. Generally, suchsubjects are “mammals”, with humans being of interest. Other subjectscan include domestic pets (e.g., dogs and cats), livestock (e.g., cows,pigs, goats, horses, and the like), rodents (e.g., mice, guinea pigs,and rats, e.g., as in animal models of disease), as well as non-humanprimates (e.g., chimpanzees, and monkeys).

The amount of polypeptide-drug conjugate administered can be initiallydetermined based on guidance of a dose and/or dosage regimen of theparent drug. In general, the polypeptide-drug conjugates can provide fortargeted delivery and/or enhanced serum half-life of the bound drug,thus providing for at least one of reduced dose or reducedadministrations in a dosage regimen. Thus, the polypeptide-drugconjugates can provide for reduced dose and/or reduced administration ina dosage regimen relative to the parent drug prior to being conjugatedin an polypeptide-drug conjugate of the present disclosure.

Furthermore, as noted above, because the polypeptide-drug conjugates canprovide for controlled stoichiometry of drug delivery, dosages ofpolypeptide-drug conjugates can be calculated based on the number ofdrug molecules provided on a per polypeptide-drug conjugate basis.

In some embodiments, multiple doses of a polypeptide-drug conjugate areadministered. The frequency of administration of a polypeptide-drugconjugate can vary depending on any of a variety of factors, e.g.,severity of the symptoms, condition of the subject, etc. For example, insome embodiments, a polypeptide-drug conjugate is administered once permonth, twice per month, three times per month, every other week (qow),once per week (qw), twice per week (biw), three times per week (tiw),four times per week, five times per week, six times per week, everyother day (qod), daily (qd), twice a day (qid), or three times a day(tid).

Methods of Treating Cancer

The present disclosure provides methods for delivering a cancerchemotherapeutic agent to an individual having a cancer. The methods areuseful for treating a wide variety of cancers, including carcinomas,sarcomas, leukemias, and lymphomas.

Carcinomas that can be treated using a subject method include, but arenot limited to, esophageal carcinoma, hepatocellular carcinoma, basalcell carcinoma (a form of skin cancer), squamous cell carcinoma (varioustissues), bladder carcinoma, including transitional cell carcinoma (amalignant neoplasm of the bladder), bronchogenic carcinoma, coloncarcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma,including small cell carcinoma and non-small cell carcinoma of the lung,adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma,breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma,sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renalcell carcinoma, ductal carcinoma in situ or bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcarcinoma, uterine carcinoma, testicular carcinoma, osteogeniccarcinoma, epithelial carcinoma, and nasopharyngeal carcinoma, etc.

Sarcomas that can be treated using a subject method include, but are notlimited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma,rhabdomyosarcoma, and other soft tissue sarcomas.

Other solid tumors that can be treated using a subject method include,but are not limited to, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Leukemias that can be treated using a subject method include, but arenot limited to, a) chronic myeloproliferative syndromes (neoplasticdisorders of multipotential hematopoietic stem cells); b) acutemyelogenous leukemias (neoplastic transformation of a multipotentialhematopoietic stem cell or a hematopoietic cell of restricted lineagepotential; c) chronic lymphocytic leukemias (CLL; clonal proliferationof immunologically immature and functionally incompetent smalllymphocytes), including B-cell CLL, T-cell CLL prolymphocytic leukemia,and hairy cell leukemia; and d) acute lymphoblastic leukemias(characterized by accumulation of lymphoblasts). Lymphomas that can betreated using a subject method include, but are not limited to, B-celllymphomas (e.g., Burkitt's lymphoma); Hodgkin's lymphoma; non-Hodgkin'sB cell lymphoma; and the like.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. By “average” is meant the arithmeticmean. Standard abbreviations may be used, e.g., bp, base pair(s); kb,kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h orhr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt,nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c.,subcutaneous(ly); and the like.

General Synthetic Procedures

Many general references providing commonly known chemical syntheticschemes and conditions useful for synthesizing the disclosed compoundsare available (see, e.g., Smith and March, March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, Fifth Edition,Wiley-Interscience, 2001; or Vogel, A Textbook of Practical OrganicChemistry, Including Qualitative Organic Analysis, Fourth Edition, NewYork: Longman, 1978).

Compounds as described herein can be purified by any purificationprotocol known in the art, including chromatography, such as HPLC,preparative thin layer chromatography, flash column chromatography andion exchange chromatography. Any suitable stationary phase can be used,including normal and reversed phases as well as ionic resins. In certainembodiments, the disclosed compounds are purified via silica gel and/oralumina chromatography. See, e.g., Introduction to Modern LiquidChromatography, 2nd Edition, ed. L. R. Snyder and J. J. Kirkland, JohnWiley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl,Springer-Verlag, N.Y., 1969.

During any of the processes for preparation of the subject compounds, itmay be necessary and/or desirable to protect sensitive or reactivegroups on any of the molecules concerned. This may be achieved by meansof conventional protecting groups as described in standard works, suchas J. F. W. McOmie, “Protective Groups in Organic Chemistry”, PlenumPress, London and New York 1973, in T. W. Greene and P. G. M. Wuts,“Protective Groups in Organic Synthesis”, Third edition, Wiley, New York1999, in “The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer),Academic Press, London and New York 1981, in “Methoden der organischenChemie”, Houben-Weyl, 4^(+h) edition, Vol. 15/1, Georg Thieme Verlag,Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, “Aminosauren, Peptide,Proteine”, Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982,and/or in Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharide andDerivate”, Georg Thieme Verlag, Stuttgart 1974. The protecting groupsmay be removed at a convenient subsequent stage using methods known fromthe art.

The subject compounds can be synthesized via a variety of differentsynthetic routes using commercially available starting materials and/orstarting materials prepared by conventional synthetic methods. A varietyof examples of synthetic routes that can be used to synthesize thecompounds disclosed herein are described in the schemes below.

HPLC Analyses

HPLC analyses were conducted on an Agilent 1100 Series Analytical HPLCequipped with a Model G1322A Degasser, Model G13 11A Quarternary Pump,Model G1329A Autosampler, Model G1314 Variable Wavelength Detector, andModel G1364C Fraction Collector at room temperature according to themethods described below.

Method A: Agilent Poroshell 120 SB C18, 4.6 mm×150 mm (2.7 um) (1.0 mLmin⁻¹). Solvent A: H₂O (0.1% HCO₂H), Solvent B: CH₃CN (0.1% HCO₂H).

Time (min) Solvent B (%) 0.0 10 15.0 100 17.5 100 18.0 10 20.5 10

Method B: Agilent Poroshell 120 SB C18, 4.6 mm×50 mm (2.7 um) (2.5 mLmin⁻¹). Solvent A: H₂O (0.1% HCO₂H), Solvent B: CH₃CN (0.1% HCO₂H)

Time (min) Solvent B (%) 0.0 10 5.0 100 6.0 100 6.1 10 7.1 10

Method C: Agilent Poroshell 120 SB C18, 4.6 mm×150 mm (2.7 um) (1.0 mLmin⁻¹). Solvent A: H₂O (0.1% HCO₂H), Solvent B: CH₃CN (0.1% HCO₂H)

Time (min) Solvent B (%) 0.0 5 15.0 95 17.5 95 18.0 5 20.5 5

Method D: Agilent Zorbax 300 SB CN, 4.6 mm×250 mm (5.0 um) (1.0 mLmin⁻¹).

Solvent A: NH₄OAc (10 mM), Solvent B: H₂O, CH₃CN (0.05, 0.95)

Time (min) Solvent B (%) 0.0 10 30.0 100 33.0 100 36.0 10 45.0 10

EXAMPLE 1 Method 1—Preparation of(2S,15S)-1-((S)-3-maytansinyl)-15-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-2,3-dimethyl-1,4,14-trioxo-7,10-dioxa-3,13-diazaoctadecan-18-oicacid (HIPS Indole E (CO₂H) PEG₂ Maytansine) (Compound 1)

Reaction schemes for the synthesis of compound HIPS Indole E (CO₂H) PEG₂Maytansine are shown in FIG. 3 and FIG. 4.

Preparation of(S)-5-(3-(tert-butoxy)-3-oxopropyl)-1-(9H-fluoren-9-yl)-3,6-dioxo-2,10,13-trioxa-4,7-diazahexadecan-16-oicacid (FMOC E (CO₂tBu) PEG₂ CO₂H) (Compound 2)(FIG. 3)

To a 20 mL glass scintillation vial containing a pea stir bar was addedH₂N PEG₂ CO₂H (710.3 mg, 4.0 mmol), Na₂CO₃ (637.9 mg, 6.0 mmol), and H₂O(10 mL). The clear, colorless solution was stirred at room temperature.N-α-Fmoc-L-glutamic acid γ-t.-butyl ester pentafluorophenyl ester(1185.7 mg, 2.0 mmol) was added to a separate 20 mL glass scintillationvial, dissolved in anhydrous 1,4-dioxane (10 mL), and added dropwise,slowly, to the aqueous solution. A white precipitate evolved withcontinued stirring. The reaction was stirred at room temperature for 4h, diluted with H₂O (70 mL), acidified to pH 3 with dropwise addition of1 M HCl, and extracted with 2×50 mL EtOAc. The organic fractions werecombined, dried over Na₂SO₄, concentrated, adsorbed onto a Biotage KPC18 HS 12 g samplet, and purified on a Biotage KP C18 HS 60 g cartridgeusing a gradient of 0-100% CH₃CN in H₂O, giving the desired product as aclear, viscous oil (1137.3 mg, 97% yield). HPLC retention time 12.260min. Method A.

Preparation of (2S,15S)-18-tert-butyl 1-((S)-3-maytansinyl)15-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2,3-dimethyl-4,14-dioxo-7,10-dioxa-3,13-diazaoctadecane-1,18-dioate(FMOC E (CO₂tBu) PEG₂Maytansine) (Compound 3)(FIG. 3)

To a dried 20 mL glass scintillation vial containing a dried flea stirbar was added FMOC E (CO₂tBu) PEG₂ CO₂H (311.3 mg, 0.5 mmol), HATU(201.0 mg, 0.5 mmol), and anhydrous DMF (2 mL). The clear, colorlesssolution was stirred at room temperature for 15 min. Deacyl maytansine(343.4 mg, 0.5 mmol), DIPEA (204.8 mg, 276.0 uL, 1.6 mmol), andanhydrous DMF (1 mL) were combined in a separate, dried 4 mL glassscintillation vial and added dropwise, slowly, to the stirring solution.The reaction was allowed to stir at room temperature for 2 h, adsorbeddirectly onto a Biotage KP C18 HS 12 g samplet, and purified on aBiotage KP C18 HS 60 g cartridge using a gradient of 0-100% CH₃CN inH₂O, giving the title compound as a white solid (377.8 mg, 59% yield).HPLC retention time 14.385 min. Method A.

Preparation of (2S,15S)-18-tert-butyl 1-((S)-3-maytansinyl)15-amino-2,3-dimethyl-4,14-dioxo-7,10-dioxa-3,13-diazaoctadecane-1,18-dioate(E (CO₂tBu) PEG₂Maytansine) (Compound 4)(FIG. 3)

To a dried 20 mL glass scintillation vial containing a dried flea stirbar was added FMOC E (CO₂tBu) PEG₂ Maytansine (377.8 mg, 0.3 mmol).Piperidine (528.8 mg, 0.613 mL, 6.2 mmol) in DMF (2.452 mL) was added bysyringe. The solution was stirred at room temperature for 20 min,adsorbed directly onto a Biotage KP C18 HS 12 g samplet, and purified ona Biotage KP C18 HS 60 g cartridge using a gradient of 0-100% CH₃CN inH₂O, giving the desired product as a pale yellow solid (269.2 mg, 87%yield). HPLC retention time 9.683 min. Method A.

Preparation of (2S,155)-18-tert-butyl 1-((S)-3-maytansinyl)15-(3-(24(2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-2,3-dimethyl-4,14-dioxo-7,10-dioxa-3,13-diazaoctadecane-1,18-dioate(FMOC HIPS Indole E (CO₂tBu) PEG₂Maytansine) (Compound 5)(FIG. 4)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added E (CO₂tBu) PEG₂ Maytansine (269.2 mg, 0.3 mmol), FMOC HIPSIndole CO₂PFP (211.5 mg, 0.3 mmol), DIPEA (105.1 mg, 141.6 uL, 0.8mmol), and anhydrous DMF (1 mL). The solution was stirred at roomtemperature for 2 h, adsorbed directly onto a Biotage KP C18 HS 3 gsamplet, and purified on a Biotage KP C18 HS 30 g cartridge using agradient of 0-100% CH₃CN in H₂O, giving the title compound as a paleyellow solid (361.6 mg, 92% yield). HPLC retention time 15.989 min.Method A.

Preparation of(2S,15S)-15-(3-(24(2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-1-((S)-3-maytansinyl)-2,3-dimethyl-1,4,14-trioxo-7,10-dioxa-3,13-diazaoctadecan-18-oicacid (FMOC HIPS Indole E (CO₂H) PEG₂ Maytansine) (Compound 6)(FIG. 4)

To a dried 20 mL glass scintillation vial containing a dried flea stirbar was added FMOC HIPS Indole E (CO₂tBu) PEG₂ Maytansine (361.6 mg, 0.2mmol) and anhydrous CH₂Cl₂ (3 mL). The solution was cooled to 0° C. inan ice, H₂O bath and SnC1₄ (1.2 mL, 1.2 mmol, 1.0 M solution inanhydrous CH₂Cl₂) was added dropwise by syringe, giving a pale yellowprecipitate. The solution was stirred at 0° C. for 5 min, whereupon theice, H₂O bath was removed and the solution warmed to room temperature.The reaction was stirred for 1 h, adsorbed directly onto a Biotage KPC18 HS 3 g samplet, and purified on a Biotage KP C18 HS 30 g cartridgeusing a gradient of 0-100% CH₃CN in H₂O, giving the desired product as apale yellow solid (260.9 mg, 75% yield). HPLC retention time 13.833 min.Method A.

Preparation of(2S,15S)-14(S)-3-maytansinyl)-15-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-2,3-dimethyl-1,4,14-trioxo-7,10-dioxa-3,13-diazaoctadecan-18-oicacid (HIPS Indole E (CO₂H) PEG₂ Maytansine) (Compound 7) (FIG. 4)

To a dried 20 mL glass scintillation vial containing a dried flea stirbar was added FMOC HIPS Indole E (CO₂H) PEG₂ Maytansine (260.9 mg, 0.2mmol). Piperidine (344.8 mg, 0.4 mL, 4.0 mmol) in DMA (1.6 mL) was addedby syringe. The solution was stirred at room temperature for 20 min,adsorbed directly onto a Biotage KP C18 HS 3 g samplet, and purified ona Biotage KP C18 HS 30 g cartridge using a gradient of 0-100% CH₃CN inH₂O, giving the title compound as a white solid (218.1 mg, 99% yield).

HPLC retention time 9.372 min. Method A. LRMS (ESI) calcd forC₅₈H₈₂ClN₈O₁₆ [M+H]: 1181.6 found 1181.3.

EXAMPLE 2 Method 2—Preparation of(2S,15S)-14(S)-3-maytansinyl)-15-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-2,3-dimethyl-1,4,14-trioxo-7,10-dioxa-3,13-diazaoctadecan-18-oicacid (HIPS Indole E (CO₂H) PEG₂ Maytansine) (Compound 8)

Reaction schemes for the synthesis of compound HIPS Indole E (CO₂H) PEG₂Maytansine are shown in FIG. 5 and FIG. 6.

Preparation of(S)-5-(3-(tert-butoxy)-3-oxopropyl)-1-(9H-fluoren-9-yl)-3,6-dioxo-2,10,13-trioxa-4,7-diazahexadecan-16-oicacid (FMOG E (CO₂tBu) PEG₂CO₂H)(FIG. 5)

Prepared as in Method 1. HPLC retention time 12.260 min. Method A.

Preparation of(S)-7-amino-2,2-dimethyl-4,8-dioxo-3,12,15-trioxa-9-azaoctadecan-18-oicacid (E (CO₂tBu) PEG₂ CO₂H) (Compound 8) (FIG. 5)

To a dried 20 mL glass scintillation vial containing a dried flea stirbar was added FMOC E (CO₂tBu) PEG₂ CO₂H (2638.7 mg, 4.5 mmol).Piperidine (1921.4 mg, 2.229 mL, 22.6 mmol) in DMF (8.871 mL) was addedby syringe. The solution was stirred at room temperature for 20 min,giving a large amount of white precipitate. The reaction was filtered,adsorbed directly onto a Biotage KP C18 HS 12 g samplet (×2), andpurified on a Biotage KP C18 HS 60 g cartridge (×2) using a gradient of0-100% CH₃CN in H₂O, giving the desired product as a colorless, viscousoil (813.1 mg, 50% yield). HPLC retention time 5.480 min. Method A.

Preparation of(S)-7-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-2,2-dimethyl-4,8-dioxo-3, 12,15-trioxa-9-azaoctadecan-18-oic acid (FMOC HIPS Indole E (CO₂tBu)PEG₂CO₂H) (Compound 9) (FIG. 5)

To a 20 mL glass scintillation vial containing a pea stir bar was addedE (CO₂tBu) PEG₂ CO₂H (582.4 mg, 1.6 mmol), FMOC HIPS Indole CO₂PFP(1253.7 mg, 1.9 mmol), DIPEA (623.1 mg, 839.8 uL, 4.8 mmol), andanhydrous DMF (5 mL). The solution was stirred at room temperature for 2h, adsorbed directly onto a Biotage KP C18 HS 12 g samplet, and purifiedon a Biotage KP C18 HS 60 g cartridge using a gradient of 0-100% CH₃CNin H₂O, giving the title compound as a waxy solid (406.3 mg, 31% yield).HPLC retention time 4.037 min. Method B.

Preparation of (2S,15S)-18-tert-butyl 1-((S)-3-maytansinyl)15-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-2,3-dimethyl-4,14-dioxo-7,10-dioxa-3,13-diazaoctadecane-1,18-dioate(FMOC HIPS Indole E (CO₂tBu) PEG₂Maytansine) (Compound 10) (FIG. 6)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC HIPS Indole E (CO₂tBu) PEG₂CO₂H (289.8 mg, 0.4 mmol),HATU (133.7 mg, 0.4 mmol), and anhydrous DMF (1 mL). The clear,colorless solution was stirred at room temperature for 15 min. Deacylmaytansine (227.5 mg, 0.4 mmol), DIPEA (135.7 mg, 182.9 uL, 1.1 mmol),and anhydrous DMF (1 mL) were combined in a separate, dried 4 mL glassscintillation vial and added dropwise, slowly, to the stirring solution.The reaction was allowed to stir at room temperature for 2 h, adsorbeddirectly onto a Biotage KP C18 HS 3 g samplet, and purified on a BiotageKP C18 HS 30 g cartridge using a gradient of 0-100% CH₃CN in H₂O, givingthe desired product as a white solid (268.4 mg, 53% yield). HPLCretention time 13.833 min. Method A.

Preparation of(2S,15S)-15-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-1-((S)-3-maytansinyl)-2,3-dimethyl-1,4,14-trioxo-7,10-dioxa-3,13-diazaoctadecan-18-oicacid (FMOC HIPS Indole E (CO₂H) PEG₂ Maytansine) (FIG. 6)

Prepared as in Method 1. HPLC retention time 13.833 min. Method A.

Preparation of (2S,15S)-1-((S)-3-maytansinyl)-15-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-2,3-dimethyl-1,4,14-trioxo-7,10-dioxa-3,13-diazaoctadecan-18-oicacid (HIPS Indole E (CO₂H) PEG₂ Maytansine) (FIG. 6)

Prepared as in Method 1. HPLC retention time 9.372 min. Method A. LRMS(ESI) calcd for C₅₈H₈₂ClN₈O₁₆ [M+H]⁺: 1181.6 found 1181.3.

EXAMPLE 3 Method 3—Preparation of (2S,15S)-1-((S)-3-maytansinyl)15-(2-amino-2-oxoethyl)-19-(24(1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-2,3-dimethyl-4,14,17-trioxo-7,10-dioxa-3,13,16-triazanonadecan-1-oate(HIPS Indole N (CONH₂) PEG₂ Maytansine) (Acidic Deprotection)

Reaction schemes for the synthesis of compound HIPS Indole N (CONH₂)PEG₂ Maytansine are shown in FIG. 7 and FIG. 8.

Preparation of (5)-tert-butyl1-(9H-fluoren-9-yl)-3,6-dioxo-5-(2-oxo-2-(tritylamino)ethyl)-2,10,13-trioxa-4,7-diazahexadecan-16-oate(FMOC N (Trt) PEG₂CO₂tBu) (Compound 11)(FIG. 7)

To a dried 20 mL glass scintillation vial containing a dried pea stirbar was added FMOC N (Trt) CO₂H (597.0 mg, 1.0 mmol), HATU (380.9 mg,1.0 mmol), and anhydrous DMF (3 mL). The clear, colorless solution wasstirred at room temperature for 15 min. H₂N PEG₂ CO₂tBu (238.2 mg, 1.0mmol), DIPEA (258.5 mg, 348.4 uL, 2.0 mmol), and anhydrous DMF (3 mL)were combined in a separate, dried 4 mL glass scintillation vial andadded dropwise, slowly, to the stirring solution. The reaction wasstirred at room temperature for 2 h, added to H₂O (100 mL) and 5 M NaCl(25 mL) in a separatory funnel, and extracted with 5×25 mL EtOAc. Theorganic fractions were combined, washed with 1×25 mL H₂O, 1×25 mL 1.2 MNaHCO₃, and 1×25 mL 5 M NaCl, dried over Na₂SO₄, concentrated, adsorbedonto a Biotage KP C18 HS 12 g samplet, and purified on a Biotage KP C18HS 60 g cartridge using a gradient of 0-100% CH₃CN in H₂O, giving thetitle compound as a white, crystalline solid (708.4 mg, 87% yield). HPLCretention time 15.971 min. Method A.

Preparation of(S)-5-(2-amino-2-oxoethyl)-1-(9H-fluoren-9-yl)-3,6-dioxo-2,10,13-trioxa-4,7-diazahexadecan-16-oicacid (FMOC N (CONH₂) PEG₂ CO₂H) (Compound 12) (FIG. 7)

To a 20 mL glass scintillation vial containing a pea stir bar was addeda solution of TFA, TIPS, H₂O (6.8 ml, 0.2 mL, 0.2 mL). FMOC N (Trt) PEG₂CO₂tBu (708.4 mg, 0.9 mmol) was added in small portions and the reactionwas stirred at room temperature for 4 h. The solution was evaporated,adsorbed onto a Biotage KP C18 HS 12 g samplet, and purified on aBiotage KP C18 HS 60 g cartridge using a gradient of 0-100% CH₃CN inH₂O, giving the desired product as a white solid (385.3 mg, 86% yield).HPLC retention time 8.084 min. Method A.

Preparation of (5S,18S)-1-((S)-3-maytansinyl)5-(2-amino-2-oxoethyl)-1-(9H-fluoren-9-yl)-17,18-dimethyl-3,6,16-trioxo-2,10,13-trioxa-4,7,17-triazanonadecan-19-oate(FMOC N (CONH₂) PEG₂Maytansine) (Compound 13) (FIG. 7)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC N (CONH₂) PEG₂ CO₂H (98.6 mg, 0.2 mmol), HATU (72.9mg, 0.2 mmol), and anhydrous DMF (1 mL). The clear, colorless solutionwas stirred at room temperature for 15 min. Deacyl maytansine (122.1 mg,0.2 mmol), DIPEA (72.8 mg, 98.1 uL, 0.6 mmol), and anhydrous DMF (1 mL)were combined in a separate, dried 4 mL glass scintillation vial andadded dropwise, slowly, to the stirring solution. The reaction wasallowed to stir at room temperature for 2 h, adsorbed directly onto aBiotage KP C18 HS 3 g samplet, and purified on a Biotage KP C18 HS 30 gcartridge using a gradient of 0-100% CH₃CN in H₂O, giving the titlecompound as a white solid (149.1 mg, 69% yield). HPLC retention time11.569 min. Method A.

Preparation of (2S,15S)-1-((S)-3-maytansinyl)15,17-diamino-2,3-dimethyl-4,14,17-trioxo-7,10-dioxa-3,13-diazaheptadecan-1-oate(N (CONH₂) PEG₂Maytansine) (Compound 14) (FIG. 8)

To a dried 20 mL glass scintillation vial containing a dried flea stirbar was added FMOC N (CONH₂) PEG₂ Maytansine (149.1 mg, 0.1 mmol).Piperidine (221.6 mg, 0.26 mL) in DMF (1.03 mL) was added by syringe.The solution was stirred at room temperature for 20 min, adsorbeddirectly onto a Biotage KP C18 HS 3 g samplet, and purified on a BiotageKP C18 HS 30 g cartridge using a gradient of 0-100% CH₃CN in H₂O, givingthe desired product as a white solid (115.0 mg, 96% yield). HPLCretention time 8.154 min. Method A.

Preparation of (2S,15S)-1-((S)-3-maytansinyl)19-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-15-(2-amino-2-oxoethyl)-2,3-dimethyl-4,14,17-trioxo-7,10-dioxa-3,13,16-triazanonadecan-1-oate(FMOC HIPS Indole N (CONH₂) PEG₂ Maytansine) (Compound 15) (FIG. 8)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added N (CONH₂) PEG₂ Maytansine (115.0 mg, 0.1 mmol), FMOC HIPSIndole CO₂PFP (86.1 mg, 0.1 mmol), DIPEA (32.3 mg, 43.5 uL, 0.3 mmol),and anhydrous DMF (1 mL). The solution was stirred at room temperaturefor 2 h, adsorbed directly onto a Biotage KP C18 HS 3 g samplet, andpurified on a Biotage KP C18 HS 30 g cartridge using a gradient of0-100% CH₃CN in H₂O, giving the title compound as a very pale yellowsolid (143.3 mg, 83% yield). HPLC retention time 13.557 min. Method A.

Preparation of(2S,15S)-1-((S)-3-maytansinyl)15-(2-amino-2-oxoethyl)-19-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-2,3-dimethyl-4,14,17-trioxo-7,10-dioxa-3,13,16-triazanonadecan-1-oate(HIPS Indole N (CONH₂) PEG₂ Maytansine) (Compound 16) (FIG. 8)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC HIPS Indole N (CONH₂) PEG₂ Maytansine (83.3 mg, 0.1mmol). Piperidine (102.1 mg, 0.118 mL, 1.2 mmol) in DMA (0.474 mL) wasadded by syringe. The solution was stirred at room temperature for 20min, adsorbed directly onto a Biotage KP C18 HS 1.2 g samplet, andpurified on a Biotage KP C18 HS 12 g cartridge using a gradient of0-100% CH₃CN in H₂O, giving the desired product as a white solid (38.8mg, 55% yield). HPLC retention time 9.084 min. Method A. LRMS (ESI)calcd for C₅₇H₈₀ClN₉NaO₁₅ [M+Na]⁺: 1188.5 found 1188.5.

EXAMPLE 4 Method 4—Preparation of HIPS Indole Cadaverine Alexa Fluor 555

A reaction scheme for the synthesis of compound HIPS Indole CadaverineAlexa Fluor 555 is shown in FIG. 9.

Preparation of (9H-fluoren-9-yl)methyl2-((1-(3-((5-(carboxamido)pentyl)amino)-3-oxopropyl)-1H-indol-2-yl)methyl)-1,2-dimethylhydrazinecarboxylateAlexa Fluor 555 (FMOC HIPS Indole Cadaverine Alexa Fluor 555) (Compound17) (FIG. 9)

To a 4 mL glass scintillation vial containing a flea stir bar was addedAlexa Fluor 555 Cadaverine (8 mg, 0.01 mmol) in DMF, H₂O (285 uL, 15uL), FMOC HIPS Indole CO₂PFP (36.1 mg, 0.06 mmol), and D1PEA (5.4 mg,7.3 uL, 0.04 mmol). The reaction was stirred at room temperature for 2h, adsorbed directly onto a Biotage KP C18 HS 1.2 g samplet, andpurified on a Biotage KP C18 HS 12 g cartridge using a gradient of0-100% CH₃CN in H₂O, giving the title compound as a purplish red film(11.8 mg, 99% yield). HPLC retention time 6.092 min. Method A.

Preparation ofN-(5-carboxamidopentyl)-3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamideAlexa Fluor 555 (HIPS Indole Cadaverine Alexa Fluor 555) (Compound18)(FIG. 9)

To a 4 mL glass scintillation vial containing a flea stir bar was addedFMOC HIPS Indole Cadaverine Alexa Fluor 555 (22.1 mg, 0.02 mmol).Piperidine (51.7 mg, 0.06 mL, 0.6 mmol) in DMA, H₂O (228 uL, 12 uL) wasadded by syringe. The solution was stirred at room temperature for 20min, adsorbed directly onto a Biotage KP C18 HS 1.2 g samplet, andpurified on a Biotage KP C18 HS 12 g cartridge using a gradient of0-100% CH₃CN in H₂O, giving the desired product as a purplish red film(18.4 mg, 99% yield). HPLC retention time 5.085 min. Method A.

EXAMPLE 5 Method 5—Preparation of(S)-1-carboxamido-13-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-1,12-dioxo-5,8-dioxa-2,11-diazahexadecan-16-oicacid ATTO 550 (HIPS Indole E (CO₂H) PEG₂ NH ATTO 550)

Reaction schemes for the synthesis of compound HIPS Indole E (CO₂H) PEG₂NH ATTO 550 are shown in FIG. 10 and FIG. 11.

Preparation of (S)-tert-butyl16-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2,2-dimethyl-4,15-dioxo-3,8,11-trioxa-5,14-diazanonadecan-19-oate(FMOC E (CO₂tBu) PEG₂ NHBOC) (Compound 19)(FIG. 10)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added N-α-Fmoc-L-glutamic acid γ-tert-butyl esterpentafluorophenyl ester (296.2 mg, 0.5 mmol), H₂N PEG₂ NHBOC (124.5 mg,0.5 mmol), DIPEA (129.2 mg, 174.2 uL, 1.0 mmol), and anhydrous DMF (0.5mL). The clear, colorless solution was stirred at room temperature for 2h, diluted with H₂O (8 mL) and 5 M NaCl (2 mL), and extracted with 3×5mL EtOAc. The organic fractions were washed with 1×5 mL H₂O, 1×5 mL 1.2M NaHCO₃, and 1×5 mL 5 M NaCl, dried over Na₂SO₄, concentrated, adsorbedonto a Biotage KP C18 HS 3 g samplet, and purified on a Biotage KP C18HS 30 g cartridge using a gradient of 0-100% CH₃CN in H₂O, giving thetitle compound as a white, crystalline solid (253.9 mg, 77% yield). HPLCretention time 14.333 min. Method A.

Preparation of (S)-tert-butyl16-amino-2,2-dimethyl-4,15-dioxo-3,8,11-trioxa-5,14-diazanonadecan-19-oate(E (CO₂tBu) PEG₂ NHBOC) (Compound 20) (FIG. 10)

To a dried 20 mL glass scintillation vial containing a dried flea stirbar was added FMOC E (CO₂tBu) PEG₂ NHBOC (253.9 mg, 0.4 mmol).Piperidine (659.4 mg, 0.765 mL 7.7 mmol) in DMF (3.1 mL) was added bysyringe. The solution was stirred at room temperature for 20 min,adsorbed directly onto a Biotage KP C18 HS 12 g samplet, and purified ona Biotage KP C18 HS 60 g cartridge using a gradient of 0-100% CH₃CN inH₂O, giving the desired product as a colorless solid (165.7 mg, 99%yield). HPLC retention time 7.599 min. Method A.

Preparation of (S)-tert-butyl16-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-2,2-dimethyl-4,15-dioxo-3,8,11-trioxa-5,14-diazanonadecan-19-oate(FMOC HIPS Indole E (CO₂tBu) PEG₂ NHBOC) (Compound 21) (FIG. 10)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added E (CO₂tBu) PEG₂ NHBOC (64.8 mg, 0.15 mmol), FMOC HIPSIndole CO₂PFP (98.4 mg, 0.15 mmol), DIPEA (58.0 mg, 78.2 uL, 0.45 mmol),and anhydrous DMF (0.3 mL). The solution was stirred at room temperaturefor 2 h, adsorbed directly onto a Biotage KP C18 HS 1.2 g samplet, andpurified on a Biotage KP C18 HS 12 g cartridge using a gradient of0-100% CH₃CN in H₂O, giving the title compound as a colorless solid(83.4 mg, 62% yield). HPLC retention time 16.054 min. Method A.

Preparation of(S)-4-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-5-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-5-oxopentanoicacid (FMOC HIPS Indole E (CO₂H) PEG₂ NH₂) (Compound 22) (FIG. 11)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC HIPS Indole E (CO₂tBu) PEG₂NHBOC (83.4 mg, 0.1 mmol)and anhydrous CH₂Cl₂ (1 mL). The solution was cooled to 0° C. in an ice,H₂O bath and SnCl₄ (0.928 mL, 0.928 mmol, 1.0 M solution in anhydrousCH₂Cl₂) was added dropwise by syringe, giving a pale yellow precipitate.The solution was stirred at 0° C. for 5 min, whereupon the ice, H₂O bathwas removed and the solution warmed to room temperature. The reactionwas stirred for 1 h, adsorbed directly onto a Biotage KP C18 HS 3 gsamplet, and purified on a Biotage KP C18 HS 30 g cartridge using agradient of 0-100% CH₃CN in H₂O, giving the desired product as a whitefilm (36.2 mg, 52% yield). HPLC retention time 10.380 min. Method A.

Preparation of(S)-13-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-1-carboxamido-1,12-dioxo-5,8-dioxa-2,11-diazahexadecan-16-oicacid ATTO 550 (FMOC HIPS Indole E (CO₂H) PEG₂ NH ATTO 550) (Compound 23)(FIG. 11)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC HIPS Indole E (CO₂H) PEG₂ NH₂ (5.3 mg, 7.1 umol),ATTO 550 NHS (5.0 mg, 6.3 umol), DIPEA (2.5 mg, 3.4 uL, 19.3 umol), andanhydrous DMF (0.3 mL). The solution was stirred at room temperature for2 h, adsorbed directly onto a Biotage SNAP Ultra 1 g samplet, andpurified on a Biotage SNAP Ultra 10 g cartridge using a step gradient of0% MeOH (0.1% AcOH) in CH₂Cl₂ (0.1% AcOH) to 10% MeOH (0.1% AcOH) inCH₂Cl₂ (0.1% AcOH), giving the title compound as a purplish red film(4.9 mg, 54% yield). TLC R_(f) (SiO₂) 0.308 (10% MeOH, 0.1% AcOH, 89.9%CH₂Cl₂).

Preparation of(S)-1-carboxamido-13-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-1,12-dioxo-5,8-dioxa-2,11-diazahexadecan-16-oicacid ATTO 550 (HIPS Indole E (CO₂H) PEG₂ NH ATTO 550) (Compound 24)(FIG. 11)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC HIPS Indole E (CO₂H) PEG₂ NH ATTO 550 (4.9 mg, 3.4umol). Piperidine (51.7 mg, 60.0 uL, 0.6 mmol) in DMA (240 uL) was addedby syringe. The solution was stirred at room temperature for 20 min,adsorbed directly onto a Biotage KP C18 HS 1.2 g samplet, and purifiedon a Biotage KP C18 HS 12 g cartridge using a gradient of 0-100% CH₃CNin H₂O, giving the desired product as a dark red film (3.2 mg, 78%yield). HPLC retention time 10.568 min, 10.689 min, 10.947 min (mixtureof cadaverine isomers).

EXAMPLE 6 Method 6—Preparation of(R)-1-carboxamido-13-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-1,12-dioxo-5,8-dioxa-2,11-diazatetradecane-14-sulfonicacid ATTO 550 (HIPS Indole C (SO₃H) PEG₂ NH ATTO 550)

Reaction schemes for the synthesis of compound HIPS Indole C (SO₃H) PEG₂NH ATTO 550 are shown in FIG. 12 and FIG. 13.

Preparation of(R)-16-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2,2-dimethyl-4,15-dioxo-3,8,11-trioxa-5,14-diazaheptadecane-17-sulfonicacid (FMOC C (SO₃H) PEG₂ NHBOC) (FIG. 12)

To a dried 20 mL glass scintillation vial containing a dried pea stirbar was added FMOC C (SO₃H) CO₂H (361.0 mg, 0.9 mmol), HATU (403.0 mg,1.1 mmol), NITA (252.0 mg, 339.6 uL, 2.0 mmol), and anhydrous DMF (10mL). The clear, colorless solution was stirred at room temperature for15 min. H₂N PEG₂ NHBOC (258.0 mg, 1.0 mmol) and anhydrous DMF (1 mL)were combined in a separate, dried 4 mL glass scintillation vial andadded dropwise, slowly, to the stirring solution. The reaction wasstirred at room temperature for 16 h, concentrated, adsorbed onto aBiotage KP C18 HS 12 g samplet, and purified on a Biotage KP C18 HS 100g cartridge using a gradient of 0-100% CH₃CN in H₂O, giving the titlecompound as a colorless, glassy solid (262.0 mg, 46% yield). HPLCretention time 8.961 min. Method A.

Preparation of(R)-16-amino-2,2-dimethyl-4,15-dioxo-3,8,11-trioxa-5,14-diazaheptadecane-17-sulfonicacid (C (SO₃H) PEG₂ NHBOC) (FIG. 12)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC C (SO₃H) PEG₂ NHBOC (248.0 mg, 0.4 mmol). Piperidine(681.2 mg, 0.790 mL 8.0 mmol) in DMF (2.0 mL) was added by syringe. Thesolution was stirred at room temperature for 1.5 h, concentrated,adsorbed directly onto a Biotage KP C18 HS 3 g samplet, and purified ona Biotage KP C18 HS 30 g cartridge using a gradient of 0-100% CH₃CN inH₂O, giving the desired product as a white solid (250.0 mg, 31% yield).HPLC retention time 2.311 min. Method A.

Preparation of(R)-16-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-2,2-dimethyl-4,15-dioxo-3,8,11-trioxa-5,14-diazaheptadecane-17-sulfonicacid (FMOC HIPS Indole C (SO₃H) PEG₂ NHBOC) (FIG. 12)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added C (SO₃H) PEG₂ NHBOC (50.0 mg, 0.13 mmol), FMOC HIPS IndoleCO₂PFP (85.0 mg, 0.13 mmol), NaHCO₃ (51.0 mg, 0.61 mmol), and DMA (2.0mL). The solution was stirred at room temperature for 1.5 h, adsorbeddirectly onto a Biotage KP C18 HS 3 g samplet, and purified on a BiotageKP C18 HS 30 g cartridge using a gradient of 0-100% CH₃CN in H₂O, givingthe title compound as a white, foamy solid (41.0 mg, 36% yield). HPLCretention time 11.024 min. Method A.

Preparation of(R)-2-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-3-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-3-oxopropane-1-sulfonicacid (FMOC HIPS Indole C (SO₃H) PEG₂NH₂) (FIG. 13)

To a dried 20 mL glass scintillation vial containing a dried flea stirbar was added FMOC HIPS Indole C (SO₃H) PEG₂ NHBOC (41.0 mg, 0.05 mmol)and anhydrous CH₂Cl₂ (5 mL). The solution was cooled to 0° C. in an ice,H₂O bath and TFA (273.6 mg, 0.184 mL, 2.4 mmol) was added dropwise bysyringe. The solution was stirred at 0° C. for 2 h, adsorbed directlyonto a Biotage SNAP Ultra 3 g samplet, and purified on a Biotage SNAPUltra 25 g cartridge using 10% MeOH in CH₂Cl₂, giving the desiredproduct as a white solid (31.0 mg, 86% yield). HPLC retention time10.284 min. Method A.

Preparation of(R)-13-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-1-carboxamido-1,12-dioxo-5,8-dioxa-2,11-diazatetradecane-14-sulfonicacid ATTO 550 (FMOC HIPS Indole C (SO₃H) PEG₂ NH ATTO 550) (Compound 25)(FIG. 13)

To a 4 mL glass scintillation vial containing a flea stir bar was addedFMOC HIPS Indole C (SO₃H) PEG₂ NH₂ (5.3 mg, 7.0 umol), ATTO 550 NHS (5.0mg, 6.3 umol), DIPEA (4.0 mg, 5.5 uL, 31.6 umol), DMF (270.0 uL), andH₂O (30.0 uL). The solution was stirred at room temperature for 2 h,adsorbed directly onto a Biotage SNAP Ultra 1 g samplet, and purified ona Biotage SNAP Ultra 10 g cartridge using a step gradient of 0% MeOH(0.1% AcOH) in CH₂Cl₂ (0.1% AcOH) to 10% MeOH (0.1% AcOH) in CH₂Cl₂(0.1% AcOH), giving the title compound as a purplish red film (10.0 mg,99% yield). HPLC retention time 15.220 min, 15.366 min, 15.573 min(mixture of isomers). Method A.

Preparation of(R)-1-carboxamido-13-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-1,12-dioxo-5,8-dioxa-2,11-diazatetradecane-14-sulfonicacid ATTO 550 (HIPS Indole C (SO₃H) PEG₂ NH ATTO 550) (Compound 26)(FIG. 13)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC HIPS Indole C (SO₃H) PEG₂ NH ATTO 550 (10.0 mg, 6.3umol). Piperidine (51.7 mg, 60.0 uL, 0.6 mmol) in DMA (240 uL) was addedby syringe. The solution was stirred at room temperature for 20 min,adsorbed directly onto a Biotage KP C18 HS 1.2 g samplet, and purifiedon a Biotage KP C18 HS 12 g cartridge using a gradient of 0-100% CH₃CNin H₂O, giving the desired product as a dark red film (3.3 mg, 38%yield). HPLC retention time 11.343 min, 11.499 min, 11.771 min. MethodA.

EXAMPLE 7 Method 7Preparation of (S)-1-((S)-3-maytansinyl)2-(4-((((4-((2S,5S)-34-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-5-isopropyl-4,7,29,32-tetraoxo-2-(3-ureidopropyl)-10,13,16,19,22,25-hexaoxa-3,6,28,31-tetraazatetratriacontanamido)benzypoxy)carbonyl)(methyl)amino)-N-methylbutanamido)propanoate(HIPS Indole G PEG₆ Val Cit PABC NMC₃ Maytansine)

Reaction schemes for the synthesis of compound HIPS Indole G PEG₆ ValCit PABC NMC₃ Maytansine are shown in FIGS. 14 to 19; where NMC₃represents the group —N(CH₃)—(CH₂)₃ ⁻.

Preparation of tert-butyl1-(9H-fluoren-9-yl)-3,6-dioxo-2,10,13,16,19,22,25-heptaoxa-4,7-diazaoctacosan-28-oate(FMOG G PEG₆CO₂tBu) (Compound 27)

To a dried 20 mL glass scintillation vial containing a dried pea stirbar was added FMOC G CO₂H (297.6 mg, 1.0 mmol), HATU (381.0 mg, 1.0mmol), and anhydrous DMF (3 mL). The clear, colorless solution wasstirred at room temperature for 15 min. H₂N PEG₆ CO₂tBu (415.7 mg, 1.0mmol), DIPEA (387.7 mg, 522.5 uL, 3.0 mmol), and anhydrous DMF (1 mL)were combined in a separate, dried 4 mL glass scintillation vial andadded dropwise, slowly, to the stirring solution. The reaction wasstirred at room temperature for 2 h, adsorbed directly onto a Biotage KPC18 HS 12 g samplet, and purified on a Biotage KP C18 HS 60 g cartridgeusing a gradient of 0-100% CH₃CN in H₂O, giving the title compound as apale yellow, viscous oil (669.0 mg, 97% yield). HPLC retention time12.481 min. Method A.

Preparation of1-(9H-fluoren-9-yl)-3,6-dioxo-2,10,13,16,19,22,25-heptaoxa-4,7-diazaoctacosan-28-oicacid (FMOC G PEG₆CO₂H) (Compound 28) (FIG. 14)

To a 20 mL glass scintillation vial containing a pea stir bar was addeda solution of TFA, TIPS, H₂O (7.6 ml, 0.2 mL, 0.2 mL). FMOC G PEG₆CO₂tBu (669.0 mg, 1.0 mmol) was added in small portions and the reactionwas stirred at room temperature for 1 h. The solution was evaporated,adsorbed onto a Biotage KP C18 HS 12 g samplet, and purified on aBiotage KP C18 HS 60 g cartridge using a gradient of 0-100% CH₃CN inH₂O, giving the desired product as a white solid (500.2 mg, 79% yield).HPLC retention time 9.686 min. Method A.

Preparation of(S)-1-((S)-3-maytansinyl)2-(4-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-N-methylbutanamido)propanoate(FMOC NMC₃ Maytansine) (Compound 29)(FIG. 14)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC N methyl Tamino butyric acid (76.3 mg, 0.2 mmol),HATU (85.4 mg, 0.2 mmol), and anhydrous DMF (0.6 mL). The clear,colorless solution was stirred at room temperature for 15 min. Deacylmaytansine (145.4 mg, 0.2 mmol), DIPEA (86.7 mg, 116.9 uL, 0.7 mmol),and anhydrous DMF (0.3 mL) were combined in a separate, dried 4 mL glassscintillation vial and added dropwise, slowly, to the stirring solution.The reaction was allowed to stir at room temperature for 2 h, adsorbeddirectly onto a Biotage KP C18 HS 3 g samplet, and purified on a BiotageKP C18 HS 30 g cartridge using a gradient of 0-100% CH₃CN in H₂O, givingthe title compound as a white solid (182.7 mg, 84% yield). HPLCretention time 14.220 min. Method A.

Preparation of(S)-1-((S)-3-maytansinyl)2-(N-methyl-4-(methylamino)butanamido)propanoate(NMC₃ Maytansine) (Compound 30)(FIG. 14)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC NMC₃ Maytansine (182.7 mg, 0.2 mmol). Piperidine(344.8 mg, 0.4 mL, 4.0 mmol) in DMF (1.6 mL) was added by syringe. Thesolution was stirred at room temperature for 20 min, adsorbed directlyonto a Biotage KP C18 HS 3 g samplet, and purified on a Biotage KP C18HS 30 g cartridge using a gradient of 0-100% CH₃CN in H₂O, giving thedesired product as a white solid (110.6 mg, 79% yield). HPLC retentiontime 13.510 min. Method A.

Preparation of(S)-1-((S)-3-maytansinyl)2-(4-((((4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)-N-methylbutanamido)propanoate(FMOC Val Cit PABC NMC₃ Maytansine) (Compound 31)(FIG. 15)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added NMC₃ Maytansine (39.8 mg, 0.05 mmol), FMOC Val Cit PABCPNO₂P (42.0 mg, 0.06 mmol), HOAt (12.8 mg, 0.09 mmol), DIPEA (20.6 mg,27.8 uL, 0.16 mmol), and anhydrous DMF (0.7 mL). The solution wasstirred at room temperature for 24 h, adsorbed directly onto a BiotageKP C18 HS 1.2 g samplet, and purified on a Biotage KP C18 HS 12 gcartridge using a gradient of 0-100% CH₃CN in H₂O, giving the titlecompound as a pale yellow solid (46.9 mg, 64% yield). HPLC retentiontime 12.702 min. Method A.

Preparation of (S)-1-((S)-3-maytansinyl)2-(4-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)-N-methylbutanamido)propanoate(Val Cit PABC NMC₃Maytansine) (Compound 32) (FIG. 15)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC Val Cit PABC NMC₃ Maytansine (46.9 mg, 0.03 mmol).Piperidine (86.2 mg, 0.1 mL, 1.0 mmol) in DMF (0.4 mL) was added bysyringe. The solution was stirred at room temperature for 20 min,adsorbed directly onto a Biotage KP C18 HS 1.2 g samplet, and purifiedon a Biotage KP C18 HS 12 g cartridge using a gradient of 0-100% CH₃CNin H₂O, giving the desired product as a white solid (31.1 mg, 79%yield). HPLC retention time 8.774 min. Method A.

Preparation of(S)-1-((S)-3-maytansinyl)2-(4-((((4-((30S,33S)-1-(9H-fluoren-9-yl)-30-isopropyl-3,6,28,31-tetraoxo-33-(3-ureidopropyl)-2,10,13,16,19,22,25-heptaoxa-4,7,29,32-tetraazatetratriacontanamido)benzyl)oxy)carbonyl)(methyl)amino)-N-methylbutanamido)propanoate(FMOC G PEG₆Val Cit PABC NMC₃ Maytansine) (Compound 33)(FIG. 16)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC G PEG₆ CO₂H (21.5 mg, 0.03 mmol), HATU (12.7 mg, 0.03mmol), and anhydrous DMF (0.2 mL). The clear, colorless solution wasstirred at room temperature for 15 min. Val Cit PABC NMC₃ Maytansine(31.1 mg, 0.03 mmol), DIPEA (10.5 mg, 14.2 uL, 0.08 mmol), and anhydrousDMF (0.2 mL) were combined in a separate, dried 4 mL glass scintillationvial and added dropwise, slowly, to the stirring solution. The reactionwas stirred at room temperature for 2 h, adsorbed directly onto aBiotage KP C18 HS 1.2 g samplet, and purified on a Biotage KP C18 HS 12g cartridge using a gradient of 0-100% CH₃CN in H₂O, giving the titlecompound as a white solid (43.6 mg, 91% yield). HPLC retention time11.305 min. Method A.

Preparation of (S)-1-((S)-3-maytansinyl)2-(4-((((4-((26S,29S)-1-amino-26-isopropyl-2,24,27-trioxo-29-(3-ureidopropyl)-6,9,12,15,18,21-hexaoxa-3,25,28-triazatriacontanamido)benzyl)oxy)carbonyl)(methyl)amino)-N-methylbutanamido)propanoate(G PEG₆Val Cit PABC NMC₃ Maytansine) (Compound 34)(FIG. 17)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC Val Cit PABC NMC₃ Maytansine (43.6 mg, 0.03 mmol).Piperidine (86.2 mg, 0.1 mL, 1.0 mmol) in DMF (0.4 mL) was added bysyringe. The solution was stirred at room temperature for 20 min,adsorbed directly onto a Biotage KP C18 HS 1.2 g samplet, and purifiedon a Biotage KP C18 HS 12 g cartridge using a gradient of 0-100% CH₃CNin H₂O, giving the desired product as a white solid (33.7 mg, 89%yield). HPLC retention time 8.817 min. Method A.

Preparation of (9H-fluoren-9-yl)methyl2-((1-((6S,9S)-1-amino-6-((4-((10S,13S)-13-((S)-3-maytansinyl)-4,9,10-trimethyl-3,8,11-trioxo-2,12-dioxa-4,9-diazatetradecyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,33,36-pentaoxo-14,17,20,23,26,29-hexaoxa-2,7,10,32,35-pentaazaoctatriacontan-38-yl)-1H-indol-2-yl)methyl)-1,2-dimethylhydrazinecarboxylate(FMOC HIPS Indole G PEG₆ Val Cit PABC NMC₃Maytansine) (Compound 35)(FIG.18)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added G PEG₆ Val Cit PABC NMC₃ Maytansine (33.7 mg, 0.02 mmol),FMOC HIPS Indole CO₂PFP (17.4 mg, 0.03 mmol), DIPEA (8.5 mg, 11.5 uL,0.07 mmol), and anhydrous DMF (0.3 mL). The solution was stirred at roomtemperature for 2 h, adsorbed directly onto a Biotage KP C18 HS 1.2 gsamplet, and purified on a Biotage KP C18 HS 12 g cartridge using agradient of 0-100% CH₃CN in H₂O, giving the title compound as a paleyellow solid (39.3 mg, 90% yield). HPLC retention time 12.900 min.Method A.

Preparation of (S)-1-((S)-3-maytansinyl)2-(4-((((4-((2S,5S)-34-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-5-isopropyl-4,7,29,32-tetraoxo-2-(3-ureidopropyl)-10,13,16,19,22,25-hexaoxa-3,6,28,31-tetraazatetratriacontanamido)benzyl)oxy)carbonyl)(methyl)amino)-N-methylbutanamido)propanoate(HIPS Indole G PEG6 Val Cit PABC NMC₃Maytansine) (Compound 36)(FIG. 19)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC HIPS Indole G PEG₆ Val Cit PABC NMC₃ Maytansine (39.3mg, 0.02 mmol). Piperidine (86.2 mg, 0.1 mL, 1.0 mmol) in DMA (0.4 mL)was added by syringe. The solution was stirred at room temperature for20 min, adsorbed directly onto a Biotage KP C18 HS 1.2 g samplet, andpurified on a Biotage KP C18 HS 12 g cartridge using a gradient of0-100% CH₃CN in H₂O, giving the desired product as a white solid (31.3mg, 90% yield). HPLC retention time 9.425 min. Method A. LRMS (ESI)calcd for C₈₇H₁₂₉ClN₁₄NaO₂₄ ⁺ [M+Na]⁺: 1811.9 found 1811.7.

EXAMPLE 8 Method 8—Preparation of3-4(S)-1-((S)-1-cyclopropylethoxy)-1-oxopropan-2-yl)(methypamino)-2-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-3-oxopropane-1-sulfonate(HIPS Indole C (SO₃H) Maytansine)

A reaction scheme for the synthesis of compound HIPS Indole C (SO₃H)Maytansine is shown in FIG. 20.

Preparation of(R)-2-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-3-sulfopropanoicacid (FMOC HIPS Indole C (SO₃H) CO₂H) (Compound 36)(FIG. 20)

To a dried 20 mL glass scintillation vial containing a dried pea stirbar was added L-cysteic acid monohydrate (411.1 mg, 2.2 mmol), FMOC HIPSIndole CO₂PFP (704.6 mg, 1.1 mmol), DIPEA (851.8 mg, 1.148 mL, 6.6mmol), and anhydrous DMF (5 mL). The solution was stirred at roomtemperature for 4 h, adsorbed directly onto a Biotage KP C18 HS 12 gsamplet, and purified on a Biotage KP C18 HS 60 g cartridge using agradient of 0-100% CH₃CN in H₂O, giving the title compound as a whitesolid (654.2 mg, 95% yield). HPLC retention time 10.725 min. Method C.

Preparation of2-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-3-(((S)-1-((S)-3-maytansinyl)-1-oxopropan-2-yl)(methyl)amino)-3-oxopropane-1-sulfonate(FMOC HIPS Indole C (SO₃H) Maytansine) (Compound 37) (FIG. 20)

To a dried 20 mL glass scintillation vial containing a dried pea stirbar was added FMOC HIPS Indole C (SO₃H) CO₂H (586.2 mg, 0.9 mmol), HATU(351.3 mg, 0.9 mmol), and anhydrous DMF (3 mL). The clear, colorlesssolution was stirred at room temperature for 15 min. Deacyl maytansine(316.9 mg, 0.5 mmol), DIPEA (388.3 mg, 523.3 uL, 3.0 mmol), andanhydrous DMF (3 mL) were combined in a separate, dried 4 mL glassscintillation vial and added dropwise, slowly, to the stirring solution.The reaction was allowed to stir at room temperature for 2 h, adsorbeddirectly onto a Biotage KP C18 HS 12 g samplet, and purified on aBiotage KP C18 HS 60 g cartridge using a gradient of 0-100% CH₃CN inH₂O, giving the desired product as a white solid (505.9 mg, 82% yield).HPLC retention time 16.493 min, 16.899 min (mixture of diastereomers).Method C.

Preparation of3-(((S)-1-((S)-3-maytansinyl)-1-oxopropan-2-yl)(methyl)amino)-2-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-3-oxopropane-1-sulfonate(HIPS Indole C (SO₃H) Maytansine) (Compound 38) (FIG. 20)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC HIPS Indole C (SO₃H) Maytansine (253.0 mg, 0.2 mmol).Piperidine (172.4 mg, 0.2 mL, 2.0 mmol) in DMA (0.8 mL) was added bysyringe. The solution was stirred at room temperature for 20 min,adsorbed directly onto a Biotage KP C18 HS 1.2 g samplet, and purifiedon a Biotage KP C18 HS 12 g cartridge using a gradient of 0-100% CH₃CNin H₂O, giving the title compound as a white solid (83.2 mg, 40% yield).HPLC retention time 13.643 min, 13.968 min (mixture of diastereomers).Method C. LRMS (ESI) calcd for C₄₉H₆₅ClN₇O₁₄S— [M−H]⁻: 1042.4 found1042.1.

EXAMPLE 9 Method 9—Preparation of (S)-1-((S)-3-maytansinyl)31-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-2,3-dimethyl-4,26,29-trioxo-7,10,13,16,19,22-hexaoxa-3,25,28-triazahentriacontan-1-oate(HIPS Indole G PEG₆ Maytansine)

Reaction schemes for the synthesis of compound HIPS Indole G PEG₆Maytansine are shown in FIG. 21 and FIG. 22.

Preparation of (S)-1-((S)-3-maytansinyl)1-(9H-fluoren-9-yl)-29,30-dimethyl-3,6,28-trioxo-2,10,13,16,19,22,25-heptaoxa-4,7,29-triazahentriacontan-31-oate(FMOC G PEG₆ Maytansine) (Compound 39)(FIG. 21)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC G PEG₆ CO₂H (62.3 mg, 0.1 mmol), HATU (37.8 mg, 0.1mmol), and anhydrous DMF (0.2 mL). The clear, colorless solution wasstirred at room temperature for 15 min. Deacyl maytansine (63.8 mg, 0.1mmol), DIPEA (38.2 mg, 51.4 uL, 0.3 mmol), and anhydrous DMF (0.2 mL)were combined in a separate, dried 4 mL glass scintillation vial andadded dropwise, slowly, to the stirring solution. The reaction wasallowed to stir at room temperature for 2 h, adsorbed directly onto aBiotage KP C18 HS 1.2 g samplet, and purified on a Biotage KP C18 HS 12g cartridge using a gradient of 0-100% CH₃CN in H₂O, giving the desiredproduct as a white solid (87.3 mg, 70% yield). HPLC retention time12.122 min. Method A.

Preparation of (S)-1-((S)-3-maytansinyl)1-amino-25,26-dimethyl-2,24-dioxo-6,9,12,15,18,21-hexaoxa-3,25-diazaheptacosan-27-oate(G PEG₆Maytansine) (Compound 40) (FIG. 21)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC G PEG₆ Maytansine (87.3 mg, 0.07 mmol). Piperidine(172.4 mg, 0.2 mL, 2.0 mmol) in DMF (0.8 mL) was added by syringe. Thesolution was stirred at room temperature for 20 min, adsorbed directlyonto a Biotage KP C18 HS 1.2 g samplet, and purified on a Biotage KP C18HS 12 g cartridge using a gradient of 0-100% CH₃CN in H₂O, giving thetitle compound as a white solid (59.1 mg, 82% yield). HPLC retentiontime 14.090 min. Method A.

Preparation of (S)-1-((S)-3-maytansinyl)31-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-2,3-dimethyl-4,26,29-trioxo-7,10,13,16,19,22-hexaoxa-3,25,28-triazahentriacontan-1-oate(FMOC HIPS Indole G PEG₆Maytansine) (Compound 41) (FIG. 21)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added G PEG₆ Maytansine (59.1 mg, 0.06 mmol), FMOC HIPS IndoleCO₂PFP (44.0 mg, 0.07 mmol), DIPEA (36.6 mg, 49.4 uL, 0.3 mmol), andanhydrous DMF (0.3 mL). The solution was stirred at room temperature for2 h, adsorbed directly onto a Biotage KP C18 HS 1.2 g samplet, andpurified on a Biotage KP C18 HS 12 g cartridge using a gradient of0-100% CH₃CN in H₂O, giving the desired product as a white solid (67.2mg, 79% yield). HPLC retention time 13.963 min. Method A.

Preparation of (S)-1-((S)-3-maytansinyl)31-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-2,3-dimethyl-4,26,29-trioxo-7,10,13,16,19,22-hexaoxa-3,25,28-triazahentriacontan-1-oate(HIPS Indole G PEG₆ Maytansine) (Compound 42)(FIG. 22)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC HIPS Indole G PEG₆ Maytansine (67.2 mg, 0.05 mmol).Piperidine (86.2 mg, 0.1 mL, 1.0 mmol) in DMA (0.4 mL) was added bysyringe. The solution was stirred at room temperature for 20 min,adsorbed directly onto a Biotage KP C18 HS 1.2 g samplet, and purifiedon a Biotage KP C18 HS 12 g cartridge using a gradient of 0-100% CH₃CNin H₂O, giving the title compound as a white solid (48.0 mg, 84% yield).HPLC retention time 9.416 min. Method A. LRMS (ESI) calcd forC₆₃H₉₄ClN₈O₁₈ ⁺ [M+H]⁺: 1285.6 found 1285.5.

EXAMPLE 10 Method 10—Preparation of (2S,15S)-1-((S)-3-maytansinyl)15-(2-(((2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyptetrahydro-2H-pyran-2-yl)amino)-2-oxoethyl)-19-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-2,3-dimethyl-4,14,17-trioxo-7,10-dioxa-3,13,16-triazanonadecan-1-oate(HIPS Indole N ((OH)₃AcNH-β-Glc) PEG₂ Maytansine)

A reaction scheme for the synthesis of compound HIPS Indole N((OH)₃AcNH-β-Glc) PEG₂ Maytansine is shown in FIG. 23.

Preparation of(2R,3S,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((2S,15S)-1-((S)-3-maytansinyl)-15-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-2,3-dimethyl-1,4,14-trioxo-7,10-dioxa-3,13-diazaheptadecanamido)tetrahydro-2H-pyran-3,4-diyldiacetate (HIPS Indole N ((Ac)₃AcNH-β-Glc) PEG₂ Maytansine) (Compound43)(FIG. 23)

Prepared as in Method 3. HPLC retention time 9.451 min. Method A. LRMS(ESI) calcd for C₇₁H₁₀₀ClN₁₀O₂₃ ⁺ [M+H]⁺: 1495.7 found 1495.4.

Preparation of (2S,15S)-1-((S)-3-maytansinyl)15-(2-(((2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)amino)-2-oxoethyl)-19-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-2,3-dimethyl-4,14,17-trioxo-7,10-dioxa-3,13,16-triazanonadecan-1-oate(HIPS Indole N ((OH)₃AcNH-β-Glc) PEG₂ Maytansine) (Compound 44)(FIG. 23)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added HIPS Indole N ((Ac)₃AcNH-β-Glc) PEG₂ Maytansine (7.2 mg,4.8 umol) and anhydrous MeOH (1.0 mL). NH₃ (1.0 mL, 2.0 mmol, 2.0 Msolution in anhydrous MeOH) was added by syringe. The solution wasstirred at room temperature for 4 h, evaporated, adsorbed directly ontoa Biotage KP C18 HS 1.2 g samplet, and purified on a Biotage KP C18 HS12 g cartridge using a gradient of 0-100% CH₃CN in H₂O, giving thedesired product as a white film (6.5 mg, 99% yield). HPLC retention time8.520 min. Method A. LRMS (ESI) calcd for C₆₅H₉₄ClN₁₀O₂₀ ⁺ [M+H]: 1369.6found 1369.5.

EXAMPLE 11 Method 11—Preparation of (2S,9S,10R)-1-((S)-3-maytansinyl)18-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-9,10-dihydroxy-2,3-dimethyl-4,8,11,16-tetraoxo-3,7,12,15-tetraazaoctadecan-1-oate(HIPS Indole Ethylenediamine Tartaric Acid (OH)₂ Beta AlanineMaytansine) (from intermediate Compound 45)

A reaction scheme for the synthesis of compound HIPS IndoleEthylenediamine Tartaric Acid (OH)₂ Beta Alanine Maytansine is shown inFIG. 24.

Preparation of benzyl tert-butyl ethane-1,2-diyldicarbamate

To a solution of tert-butyl (2-aminoethyl)carbamate (1.0 g, 6.3 mmol),Et₃N (0.94 g, 9.4 mmol), and THF (20 mL) was added CbzCl (1.2 g, 7.5mmol) over 10 min. The reaction was stirred at room temperature for 10 hand concentrated to afford a white solid, which was purified by columnchromatography on silica gel (PE: ETOAc=10:1˜3:1) to give the desiredcompound (1.5 g, 83% yield).

Preparation of 2-(((benzyloxy)carbonyl)amino)ethanaminium chloride

Benzyl tert-butyl ethane-1,2-diyldicarbamate (1.5 g, 6.3 mmol) in MeOH(10 mL) was treated with HCl/ETOAc (2 mL). The reaction was stirred atroom temperature for 10 h and concentrated to give the title compound(1.0 g, 85% yield).

Preparation of benzyl (2-aminoethyl)carbamate

To a solution of 2-(((benzyloxy)carbonyl)amino)ethanaminium chloride(1.0 g, 4.3 mmol) in water (5 mL) was added anhydrous Na₂CO₃ untillitmus testing indicated pH 9. Solvent removal afforded a residue thatwas triturated with CH₂Cl₂ and filtered. The filtrate was concentratedto afford the desired free amine (0.93 g, 99% yield).

Preparation of(2S,3R)-2,3-diacetoxy-4-((2-(((benzyloxy)carbonyl)aminotethyltamino)-4-oxobutanoicacid

To benzyl (2-aminoethyl)carbamate (100 mg, 0.52 mmol) in THF (2 mL) at0° C. was added (+)-diacetyl-L-tartaric anhydride (37 mg, 0.17 mmol).The mixture was warmed to room temperature and stirred overnight. Thesolvent was removed to afford the title compound as a pale yellow solid(212 mg, 83% yield). The compound was used in subsequent steps withoutadditional purification.

Preparation of(2S,3R)-4-((2-(((benzyloxy)carbonyl)amino)ethyl)amino)-2,3-dihydroxy-4-oxobutanoicacid

To a suspension of(2S,3R)-2,3-diacetoxy-4-((2-(((benzyloxy)carbonyl)amino)ethyl)amino)-4-oxobutanoicacid (3.3 g, 8 mmol) in H₂O (40 mL) was added potassium hydroxide (0.9g, 16 mmol). The solution was stirred at room temperature for 1.5 h andacidified to pH 5 with HCl (1 M), giving a white precipitate. The solidwas filtered, washed with water, and dried to give the title compound(1.2 g, 45% yield). The compound was used in subsequent steps withoutadditional purification.

Preparation of (9R,10S)-benzyl9,10-dihydroxy-3,8,11-trioxo-1-phenyl-2-oxa-4,7,12-triazapentadecan-15-oate

To a solution of(2S,3R)-4-((2-(((benzyloxy)carbonyl)amino)ethyl)amino)-2,3-dihydroxy-4-oxobutanoicacid (4 g, 12 mmol) in DMF (100 mL) was added HOBT (1.66 g, 12 mmol) andEDCI (2.3 g, 12 mmol). The reaction was stirred at room temperature for30 min, whereupon benzyl 3-aminopropanoate (2.1 g, 12 mmol) was added.The reaction was allowed to stir at room temperature overnight and addeddropwise to a 10% Na₂CO₃ solution, giving a white precipitate. The solidwas filtered, washed with water and dried under vacuum to afford thetitle compound (5.2 g, 78% yield).

Preparation of (9R, 10S)-benzyl9,10-bis((tert-butyldimethylsilyl)oxy)-3,8,11-trioxo-1-phenyl-2-oxa-4,7,12-triazapentadecan-15-oate

To a solution of (9R,10S)-benzyl9,10-dihydroxy-3,8,11-trioxo-1-phenyl-2-oxa-4,7,12-triazapentadecan-15-oate(5.8 g, 12 mmol) in DMF (20 mL) was added TBSCl (18 g, 0.72 mol) andimidazole (8.1 g, 0.72 mol). The reaction was stirred at roomtemperature overnight, diluted with ETOAc (200 mL), washed with 1×5 MNaCl and 2×H₂O, dried over Na₂SO₄, and concentrated to give a paleyellow viscous oil. The oil was purified by flash column chromatographyon silica gel (PE:EA=6:1˜2:1) to afford the title compound as pale pinksolid (4.9 g, 87% yield).

Preparation of3-((2S,3R)-4-((2-aminoethyl)amino)-2,3-bis((tert-butyldimethylsilyl)oxy)-4-oxobutanamido)propanoicacid (Compound 45) (FIG. 24)

To a solution of (9R,10S)-benzyl9,10-bis((tert-butyldimethylsilyl)oxy)-3,8,11-trioxo-1-phenyl-2-oxa-4,7,12-triazapentadecan-15-oate(4.0 g, 2.46 mmol) in THF (400 mL) was added 10% Pd/C (3.7 g, 50% wt).The mixture was stirred at room temperature under a double-layer H₂balloon overnight. The reaction mixture was filtered and washed withTHF. The filtrate was concentrated to afford the title compound (3.7 g,91% yield). MS: m/z (ESI+): (M+H)⁺=492.55. 1H NMR (400 MHz, CDCl3) δ4.18 (d, J=2.0 Hz, 1H), 4.15 (d, J=2.0 Hz, 1H), 3.37-3.40 (m, 2H),3.34-3.36 (m, 1H), 3.30-3.32 (m, 1H), 2.74 (s, 2H), 2.17-2.24 (m, 2H),0.88 (s, 18H), 0.02 (s, 6H), −0.04 (s, 6H).

Preparation of3-((2S,3R)-4-((2-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)ethyl)amino)-2,3-bis((tert-butyldimethylsilyl)oxy)-4-oxobutanamido)propanoicacid (FIG. 24)

To a dried 20 mL glass scintillation vial containing a dried pea stirbar was added3-((2S,3R)-4-((2-aminoethyl)amino)-2,3-bis((tert-butyldimethylsilyl)oxy)-4-oxobutanamido)propanoicacid (236.0 mg, 0.5 mmol), FMOC HIPS Indole CO₂PFP (206.0 mg, 0.3 mmol),NaHCO₃ (134.0 mg, 1.6 mmol), and DMA (5 mL). The solution was stirred atroom temperature for 1 h, poured into H₂O (25 mL), and extracted with3×10 mL ETOAc. The organic fractions were combined, washed with 1×10 mL5 M NaCl, dried over Na₂SO₄, concentrated, adsorbed onto a Biotage KPC18 HS 3 g samplet, and purified on a Biotage KP C18 HS 30 g cartridgeusing a gradient of 0-100% CH₃CN in H₂O, giving the desired product as awhite solid (230.0 mg, 76% yield). HPLC retention time 16.21 min. MethodA.

Preparation of (2S,9S,10R)-1-((S)-3-maytansinyl)18-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-9,10-bis((tert-butyldimethylsilyl)oxy)-2,3-dimethyl-4,8,11,16-tetraoxo-3,7,12,15-tetraazaoctadecan-1-oate (FMOCHIPS Indole Ethylenediamine Tartaric Acid (OTBDMS)₂ Beta AlanineMaytansine) (FIG. 24)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added3-((2S,3R)-4-((2-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)ethyl)amino)-2,3-bis((tert-butyldimethylsilyl)oxy)-4-oxobutanamido)propanoicacid (129.0 mg, 0.1 mmol), HATU (52.0 mg, 0.1 mmol), and anhydrous DMF(0.5 mL). The clear, colorless solution was stirred at room temperaturefor 15 min. Deacyl maytansine (96.0 mg, 0.1 mmol), DIPEA (36.0 mg, 48.5uL, 0.3 mmol), and anhydrous DMF (0.5 mL) were combined in a separate,dried 4 mL glass scintillation vial and added dropwise, slowly, to thestirring solution. The reaction was allowed to stir at room temperaturefor 2 h, adsorbed directly onto a Biotage KP C18 HS 3 g samplet, andpurified on a Biotage KP C18 HS 30 g cartridge using a gradient of0-100% CH₃CN in H₂O, giving the title compound as a white solid (103.0mg, 48% yield). HPLC retention time 17.23 min. Method A.

Preparation of (2S,9S,10R)-1-((S)-3-maytansinyl)18-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-9,10-dihydroxy-2,3-dimethyl-4, 8,11,16-tetraoxo-3,7,12,15-tetraazaoctadecan-1-oate (HIPSIndole Ethylenediamine Tartaric Acid (OH)₂ Beta Alanine Maytansine)(FIG. 24)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC HIPS Indole Ethylenediamine Tartaric Acid (OTBDMS)₂Beta Alanine Maytansine (102.0 mg, 0.06 mmol) and anhydrous THF (0.6mL). The solution was cooled to 0° C. in an ice, H₂O bath and TBAF(0.225 mL, 0.225 mmol, 1.0 M solution in anhydrous THF) was addeddropwise by syringe. The solution was stirred at 0° C. for 35 min,adsorbed directly onto a Biotage KP C18 HS 1.2 g samplet, and purifiedon a Biotage KP C18 HS 12 g cartridge using a gradient of 0-100% CH₃CNin H₂O, giving the desired product as a white solid (45.0 mg, 60%yield). HPLC retention time 8.72 min. Method A. LRMS (ESI) calcd forC₅₅H₇₆ClN₉NaO₁₅ ⁺ [M+Na]⁺: 1160.5 found 1160.5.

EXAMPLE 12 Method 12Preparation of (S)-(S)-1-cyclopropylethyl16-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-7,10-bis(2-hydroxyethyl)-2,3-dimethyl-4,8,11,14-tetraoxo-3,7,10,13-tetraazahexadecan-1-oate(HIPS Indole Glycine Dihydroxypeptoid (OH)₂ Beta Alanine Maytansine)(from intermediate Compound 46)

A reaction scheme for the synthesis of compound HIPS Indole GlycineDihydroxypeptoid (OH)₂ Beta Alanine Maytansine is shown in FIG. 25.

Preparation of 2-((tert-butyldimethylsilyl)oxy)ethanamine (Compound 1)

To a stirred solution of 2-aminoethanol (50 g, 0.82 mol), Et₃N (124 g,1.23 mol), and DMAP (2 g) in anhydrous DCM (1 L) was added TBSCl (135 g,0.9016 mol). The reaction was stirred at room temperature overnight,quenched with aqueous NH₄Cl, and extracted with DCM (×3). The combinedorganic layers were washed with H₂O, dried over MgSO₄, filtered,concentrated, and purified by flash column chromatography to give thetitle compound (30.5 g, 22% yield). 1H NMR (400 MHz, CDCl3) δ 3.6 (t,J=5.5 Hz, 2H), 2.74 (t, J=5.5 Hz, 2H), 1.36 (brs, 1H), 0.87 (s, 9H),0.04 (s, 6H).

Preparation of benzyl3-((2-((tert-butyldimethylsilyl)oxy)ethyl)amino)propanoate (Compound 2)

To a stirred solution of lithium chloride (20 mg) and Compound 1 (1.0 g,5.71 mmol) in MeOH (25 mL) and THF (25 mL) at 0° C. was added benzylacrylate (1.0 g, 6.28 mmol) dropwise over 10 min. The reaction mixturewas allowed to warm to room temperature gradually and stirred at roomtemperature overnight. The reaction mixture was concentrated underreduced pressure, extracted with ETOAc (250 mL), washed with 5 M NaCl(200 mL), dried over Na₂SO₄, and evaporated. The residue was purified byflash column chromatography (PE:EA=5:1 to 0:1) to afford compound thedesired compound (0.7 g, 37% yield).

Preparation of benzyl3-(2-bromo-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acetamido)propanoate(Compound 3)

To a solution of Compound 2 (0.7 g, 2.07 mmol) in THF (20 mL) at 0° C.under nitrogen were added Et₃N (1.2 equiv, 0.25 g, 2.49 mmol) andbromoacetyl bromide (1.2 equiv, 0.5 g, 2.49 mmol). The reaction wasstirred at 0° C. for 1 h, diluted with ETOAc (10 mL), and filtered. Thesolid was rinsed with ETOAc and the filtrate dried in vacuo to yield thecrude bromoacetyl amide. The product was purified by flash columnchromatography (PE:EA=10:1 to 5:1) to afford pure Compound 3 (0.3 g, 31%yield). MS+: 459[M+H]⁺.

Preparation ofbenzyl10-(2-((tert-butyldimethylsilyl)oxy)ethyl)-2,2,3,3-tetramethyl-9-oxo-4-oxa-7,10-diaza-3-silatridecan-13-oate(Compound 4)

To a solution of Compound 3 (36.8 g, 80.3 mmol, 1.0 eq) in THF (350 mL)at 0° C. under nitrogen were added Et₃N (16.3 g, 160.7 mmol, 2 eq) and2-((tert-butyldimethylsilyl)oxy)ethanamine (28.1 g, 160.7 mmol, 2 eq).The reaction was stirred at room temperature overnight, diluted withETOAc (100 mL), and filtered. The solid was rinsed with ETOAc and thefiltrate concentrated to give the crude amine. The product was purifiedby flash column chromatography (PE:EA=3:1) to afford Compound 4 (27 g,60% yield).

Preparation of benzyl7,10-bis(2-((tert-butyldimethylsilyl)oxy)ethyl)-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazatridecan-13-oate(Compound 5)

To a solution of Cbz-glycine (2.5 g, 12 mmol), EDCI (2.5 g, 13 mmol),and HOBT (1.75 g, 13 mmol) in DCM (20 mL) at 0° C. was added DIPEA (4.2g, 32.5 mmol). The mixture was stirred at 0° C. for 30 min. To thesolution was added Compound 4 (6 g, 11 mmol) dropwise. The reaction wasstirred at room temperature overnight, evaporated to dryness, suspendedin DCM, and filtered. The filtrate was washed with H₂O and 5 M NaCl,dried over Na₂SO₄, and concentrated. The residue was purified by flashcolumn chromatography to afford Compound 5 (3.01 g, 38% yield).

Preparation of7-(2-aminoacetyl)-10-(2-((tert-butyldimethylsilyl)oxy)ethyl)-2,2,3,3-tetramethyl-9-oxo-4-oxa-7,10-diaza-3-silatridecan-13-oicacid (Compound 46)(FIG. 25)

A mixture of Compound 5 (1.9g, 2.55 mmol) and Pd/C (500 mg) in ETOAc (50mL) in a Parr shaker was stirred under H₂ (50 psi) at room temperatureovernight. The mixture was filtered though a pad of Celite, andconcentrated to give the title compound (AB4296) (730 mg, 55% yield).LC-MS: 520 (M+1).

Preparation of7-(2-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)acetyl)-10-(2-((tert-butyldimethylsilyl)oxy)ethyl)-2,2,3,3-tetramethyl-9-oxo-4-oxa-7,10-diaza-3-silatridecan-13-oicacid (FIG. 25)

To a dried 20 mL glass scintillation vial containing a dried pea stirbar was added peptoid AB4296 (264.0 mg, 0.5 mmol), FMOC HIPS IndoleCO₂PFP (220.0 mg, 0.3 mmol), NaHCO₃ (120.0 mg, 1.4 mmol), and DMA (5mL). The solution was stirred at room temperature for 6 h, poured intoH₂O (25 mL), and extracted with 3×10 mL ETOAc. The organic fractionswere combined, washed with 1×10 mL 5 M NaCl, dried over Na₂SO₄,concentrated, adsorbed onto a Biotage KP C18 HS 3 g samplet, andpurified on a Biotage KP C18 HS 30 g cartridge using a gradient of0-100% CH₃CN in H₂O, giving the title compound as a white solid (184.0mg, 55% yield). HPLC retention time 18.88 min. Method A.

Preparation of (S)-1-((S)-3-maytansinyl)7-(2-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1-indol-1-yl)propanamido)acetyl)-10-(2-((tert-butyldimethylsilyl)oxy)ethyl)-2,2,3,3,14,15-hexamethyl-9,13-dioxo-4-oxa-7,10,14-triaza-3-silahexadecan-16-oate(FMOC HIPS Indole Glycine Dihydroxypeptoid (OTBDMS)₂ Beta AlanineMaytansine)(FIG. 25)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added7-(2-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)acetyl)-10-(2-((tert-butyldimethylsilyl)oxy)ethyl)-2,2,3,3-tetramethyl-9-oxo-4-oxa-7,10-diaza-3-silatridecan-13-oicacid (180.0 mg, 0.2 mmol), HATU (72.0 mg, 0.2 mmol), and anhydrous DMF(0.5 mL). The clear, colorless solution was stirred at room temperaturefor 15 min. Deacyl maytansine (123.0 mg, 0.2 mmol), DIPEA (51.0 mg, 68.7uL, 0.4 mmol), and anhydrous DMF (0.5 mL) were combined in a separate,dried 4 mL glass scintillation vial and added dropwise, slowly, to thestirring solution. The reaction was allowed to stir at room temperaturefor 2 h, adsorbed directly onto a Biotage KP C18 HS 3 g samplet, andpurified on a Biotage KP C18 HS 30 g cartridge using a gradient of0-100% CH₃CN in H₂O, giving the desired product as a white solid (235.0mg, 80% yield). HPLC retention time 20.21 min. Method A.

Preparation of (S)-1-((S)-3-maytansinyl)16-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-7,10-bis(2-hydroxyethyl)-2,3-dimethyl-4,8,11,14-tetraoxo-3,7,10,13-tetraazahexadecan-1-oate(HIPS Indole Glycine Dihydroxypeptoid (OH)₂ Beta AlanineMaytansine)(FIG. 25)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC HIPS Indole Glycine Dihydroxypeptoid (OTBDMS)₂ BetaAlanine Maytansine (114.0 mg, 0.07 mmol) and anhydrous THF (0.5 mL). Thesolution was cooled to 0° C. in an ice, H₂O bath and TBAF (0.3 mL, 0.3mmol, 1.0 M solution in anhydrous THF) was added dropwise by syringe.The solution was stirred at 0° C. for 1 h, adsorbed directly onto aBiotage KP C18 HS 1.2 g samplet, and purified on a Biotage KP C18 HS 12g cartridge using a gradient of 0-100% CH₃CN in H₂O, giving the titlecompound as an off-white solid (41.7 mg, 51% yield). HPLC retention time8.03 min. Method A. LRMS (ESI) calcd for C₅₇H₈₀ClN₉NaO₁₅ ⁺ [M+Na]⁺:1188.5 found 1188.4.

EXAMPLE 13 Method 13Preparation of (S)-1-((S)-3-maytansinyl)19-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-7,10,13-tris(2-hydroxyethyl)-2,3-dimethyl-4,8,11,14,17-pentaoxo-3,7,10,13,16-pentaazanonadecan-1-oate(HIPS Indole Glycine Trihydroxypeptoid (OH)₃ Beta Alanine Maytansine)(from intermediate Compound 47)

A reaction scheme for the synthesis of compound HIPS Indole GlycineTrihydroxypeptoid (OH)₃ Beta Alanine Maytansine is shown in FIG. 26.

Preparation of benzyl7-(2-bromoacetyl)-10-(2-((tert-butyldimethylsilyl)oxy)ethyl)-2,2,3,3-tetramethyl-9-oxo-4-oxa-7,10-diaza-3-silatridecan-13-oate(Compound 6)

To a solution of Compound 4 (16 g, 29 mmol) in THF (150 mL) at 0° C.under nitrogen were added Et₃N (3.5 g, 34.7 mmol) and bromoacetylbromide (7 g, 34.7 mmol), dropwise. The reaction was stirred at 0° C.for 1 h, diluted with ETOAc (100 mL) and filtered. The solid was rinsedwith ETOAc and the filtrate was concentrated to give the crudebromoacetyl amide. The product was purified by flash columnchromatography (PE:EA=10:1) to afford Compound 6 (12.6 g, 64% yield).

Preparation of benzyl10,13-bis(2-((tert-butyldimethylsilyl)oxy)ethyl)-2,2,3,3-tetramethyl-9,12-dioxo-4-oxa-7,10,13-triaza-3-silahexadecan-16-oate(Compound 7)

To a solution of Compound 6 (12.6 g, 18.5 mmol) in THF (150 mL) at 0° C.under nitrogen, were added Et₃N (3.75 g, 37 mmol) and2-((tert-butyldimethylsilyl)oxy)ethanamine (6.5 g, 37 mmol). Thereaction was stirred at room temperature overnight, diluted with ETOAc(100 mL), filtered, and the solid washed with ETOAc. The filtrate wasconcentrated in vacuo to yield the crude amine. The product was purifiedby flash column chromatography (PE:EA=3:1) to afford pure Compound 7(5.8 g, 41% yield).

Preparation of benzyl7,10,13-tris(2-((tert-butyldimethylsilyl)oxy)ethyl)-3,6,9,12-tetraoxo-1-phenyl-2-oxa-4,7,10,13-tetraazahexadecan-16-oate(Compound 8)

To a solution of Cbz-glycine (1.74 g, 8.3 mmol), EDCI (1.73 g, 9.06mmol), and HOBT (1.22 g, 9.06 mmol) in DCM (50 mL) at 0° C. was addedDIPEA (2.91 g, 22.6 mmol). The solution was stirred for 30 min. To thesolution was added Compound 7 (5.8 g, 7.54 mmol), dropwise, in DCM (5mL). The mixture was stirred at room temperature overnight,concentrated, suspended in DCM, and filtered. The filtrate was washedwith H₂O and 5 M NaCl, and the product purified by flash columnchromatography to afford pure Compound 8 (2.72 g, 38% yield). LC-MS: 960(M+1).

Preparation of7-(2-aminoacetyl)-10,13-bis(2-((tert-butyldimethylsilyl)oxy)ethyl)-2,2,3,3-tetramethyl-9,12-dioxo-4-oxa-7,10,13-triaza-3-silahexadecan-16-oicacid (Compound 47) (FIG. 26)

A mixture of Compound 8 (2.7 g, 2.81 mmol) and Pd/C (1 g) in ETOAc (60mL) in a Parr shaker was stirred under H₂ (60 psi) at room temperatureovernight. The mixture was filtered though a pad of Celite, washed withMeOH, and concentrated to give a residue that was lyophilized to affordAB4297 (1.7 g, 55% yield). LC-MS: 735.6 (M+1).

Preparation of7-(2-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)acetyl)-10,13-bis(2-((tert-butyldimethylsilyl)oxy)ethyl)-2,2,3,3-tetramethyl-9,12-dioxo-4-oxa-7,10,13-triaza-3-silahexadecan-16-oicacid (FIG. 26)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added peptoid AB4297 (50.0 mg, 0.07 mmol), FMOC HIPS IndoleCO₂PFP (38.6 mg, 0.06 mmol), NaHCO₃ (14.2 mg, 0.2 mmol), and DMA (0.4mL). The solution was stirred at room temperature for 16 h, acidified topH 2 with 1 M HCl, and extracted with 1×10 mL ETOAc. The organicfraction was washed with a mixture of 1×10 mL 1 M HCl/5 M NaCl (1/1),dried over MgSO₄, filtered, concentrated, adsorbed onto a Biotage SNAPUltra 1 g samplet, and purified on a Biotage SNAP Ultra 10 g cartridgeusing a gradient of 0-10% MeOH in CH₂Cl₂, giving the title compound as awhite solid (40.0 mg, 49% yield). HPLC retention time 7.12 min. MethodA. LRMS (ESI) calcd for C₄₇H₈₇N₇O₉Si₃ [M−H]⁻: 1198.6; found 1198.0.

Preparation of (S)-1-((S)-3-maytansinyl)7-(2-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1-indol-1-yl)propanamido)acetyl)-10,13-bis(2-((tert-butyldimethylsilyl)oxy)ethyl)-2,2,3,3,17,18-hexamethyl-9,12,16-trioxo-4-oxa-7,10,13,17-tetraaza-3-silanonadecan-19-oate(FMOC HIPS Indole Glycine Trihydroxypeptoid (OTBDMS)₃ Beta AlanineMaytansine) (FIG. 26)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added7-(2-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)acetyl)-10,13-bis(2-((tert-butyldimethylsilyl)oxy)ethyl)-2,2,3,3-tetramethyl-9,12-dioxo-4-oxa-7,10,13-triaza-3-silahexadecan-16-oicacid (28.1 mg, 0.03 mmol), deacyl maytansine (15.2 mg, 0.02 mmol), andDMA (0.13 mL). The solution was cooled to 0° C. in an ice, H₂O bathwhereupon 2,4,6-trimethylpyridine (5.7 mg, 6.2 uL, 0.05 mmol) and COMU(10.0 mg, 0.02 mmol) were added. The reaction was allowed to stir at 0°C. for 15 min, then warmed to room temperature and stirred for 3 h. Thereaction mixture was diluted with ETOAc (7 mL), washed with 3×2 mL 0.5 MHCl, 3×2mL 1.2 M NaHCO₃, and 3×2 mL 5 M NaCl, dried over MgSO₄,filtered, and concentrated to a viscous oil. The product was adsorbedonto a Biotage KP C18 HS 1.2 g samplet and purified on a Biotage KP C18HS 12 g cartridge using a gradient of 0-100% CH₃CN in H₂O, giving thedesired product as a white solid (11.7 mg, 32% yield). HPLC retentiontime 18.72 min. Method A. LRMS (ESI) calcd for C₉₄H₁₃₉ClN₁₀O₁₉Si₃[M+Na]⁺: 1853.9 found 1854.7.

Preparation of (S)-1-((S)-3-maytansinyl)19-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-7,10,13-tris(2-hydroxyethyl)-2,3-dimethyl-4,8,11,14,17-pentaoxo-3,7,10,13,16-pentaazanonadecan-1-oate(HIPS Indole Glycine Trihydroxypeptoid (OH)₃ Beta Alanine Maytansine)(FIG. 26)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC HIPS Indole Glycine Trihydroxypeptoid (OTBDMS)₃ BetaAlanine Maytansine (11.7 mg, 6.4 umol) and anhydrous THF (63.4 uL). Thesolution was cooled to 0° C. in an ice, H₂O bath and TBAF (25.0 uL, 0.25mmol, 1.0 M solution in anhydrous THF) was added dropwise bymicropipettor. The solution was stirred at 0° C. for 1 h, adsorbeddirectly onto a Biotage KP C18 HS 1.2 g samplet, and purified on aBiotage KP C18 HS 12 g cartridge using a gradient of 0-100% CH₃CN inH₂O, giving the title compound as an off-white solid (4.9 mg, 61%yield). HPLC retention time 8.50 min. Method A. LRMS (ESI) calcd forC₆₁H₈₇ClN₁₀O₁₇ [M+H]⁺: 1267.6 found 1267.3.

EXAMPLE 14 Method 14—Preparation of (S)-1-((S)-3-maytansinyl)19-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-7,10,13-tris(2-methoxyethyl)-2,3-dimethyl-4,8,11,14,17-pentaoxo-3,7,10,13,16-pentaazanonadecan-1-oate(HIPS Indole Glycine Trimethoxypeptoid Beta Alanine Maytansine) (fromintermediate Compound 48)

A reaction scheme for the synthesis of compound HIPS Indole GlycineTrimethoxypeptoid Beta Alanine Maytansine is shown in FIG. 27.

Preparation of benzyl 3-((2-methoxyethyl)amino)propanoate (Compound 1)

To a solution of 2-methoxyethanamine (0.42 g, 5.6 mmol) and lithiumchloride (20 mg) in MeOH (25 mL) and THF (25 mL) at 0° C. was addedbenzyl acrylate (1.0 g, 6.16 mmol) dropwise over 10 min. The reactionwas allowed to warm to room temperature gradually and stirred at roomtemperature overnight. The solvent was removed under reduced pressureand the resulting residue dissolved in ETOAc (250 mL). The organicfraction was washed with 5 M NaCl (200 mL), dried over Na₂SO₄, andconcentrated to give a residue. The product was purified by flash columnchromatography (PE:EA=5:1 to 0:1) to afford Compound 1 (0.95 g, 71%yield).

Preparation of benzyl 3-(2-bromo-N-(2-methoxyethyl)acetamido)propanoate(Compound 2)

To a solution of Compound 1 (0.95 g, 4 mmol) in THF (20 mL) at 0° C.were added Et₃N (0.49 g, 4.8 mmol) and bromoacetyl bromide (0.97 g, 4.8mmol). The reaction was stirred at 0° C. for 1 h, diluted with ETOAc (20mL), filtered, and washed with ETOAc. The filtrate was concentrated anddried in vacuo to yield the crude bromoacetyl amide which was purifiedby flash column chromatography (PE:EA=5:1) to afford pure Compound 2(1.15 g, 80% yield). (MS+: 358, 360).

Preparation of benzyl3-(N-(2-methoxyethyl)-2-((2-methoxyethyl)amino)acetamido)propanoate(Compound 3)

To a solution of Compound 2(1.15 g, 3.2 mmol) in THF (20 mL) at 0° C.were added Et₃N (0.647 g, 6.4 mmol) and 2-methoxyethanamine (0.48 g, 6.4mmol). The reaction was stirred overnight, diluted with ETOAc (10 mL),filtered, and washed with ETOAc. The filtrate was concentrated and driedin vacuo to yield the crude amine which was purified by flash columnchromatography (PE:EA=5:1 to 0:1) to afford pure Compound 3 (0.35 g, 31%yield). LC-MS: 353 [M+H]⁺.

Preparation of benzyl3-(2-(2-bromo-N-(2-methoxyethyl)acetamido)-N-(2-methoxyethyl)acetamido)propanoate(Compound 4)

To a solution of Compound 3 (0.35 g, 1 mmol) in THF (15 mL) at 0° C.were added Et₃N (0.121 g, 1.2 mmol) and bromoacetyl bromide (0.242 g,1.2 mmol). The reaction was at 0° C. for 1 h, diluted with ETOAc (10mL), and filtered. The solid was washed with ETOAc and the filtrateconcentrated in vacuo to give the crude bromoacetyl amide. Purificationby flash column chromatography (EA) afforded Compound 4 (0.29 g, 61%yield). LC-MS: 473, 475.

Preparation of benzyl8,11-bis(2-methoxyethyl)-7,10-dioxo-2-oxa-5,8,11-triazatetradecan-14-oate(Compound 5)

To a solution of Compound 4 (0.29 g, 0.61 mmol) in THF (10 mL) at 0° C.were added Et₃N (0.124 g, 1.23 mmol) and 2-methoxyethanamine (91 mg,1.23 mmol). The reaction was stirred at room temperature overnight,diluted with ETOAc (10 mL), and filtered. The solid was washed withETOAc and the filtrate concentrated to give the crude amine which waspurified by flash column chromatography (MeOH:EA=1:5) to afford pureCompound 5 (0.15 g, 52% yield). LC-MS: 468 (M+1).

Preparation of benzyl7,10,13-tris(2-methoxyethyl)-3,6,9,12-tetraoxo-1-phenyl-2-oxa-4,7,10,13-tetraazahexadecan-16-oate(Compound 6)

To a mixture of Cbz-glycine (181 mg, 0.87 mmol), DCM (10 mL), EDCI (200mg, 1.04 mmol) and HOBt (140 mg, 1.04 mmol) at 0° C. was added D1PEA(400 mg, 3.13 mmol). The mixture was stirred at 0° C. for 15 minwhereupon Compound 5 (400 mg, 0.87 mmol) was added. The reaction wasstirred at room temperature overnight, washed with H₂O and 5 M NaCl, anddried over Na₂SO₄. The crude product was concentrated and purified byflash column chromatography (PE:EA=1:3) to afford Compound 6 (300 mg,62% yield). LC-MS: 660 (M+1).

Preparation of5-(2-aminoacetyl)-8,11-bis(2-methoxyethyl)-7,10-dioxo-2-oxa-5,8,11-triazatetradecan-14-oicacid (Compound 48)(FIG. 27)

A mixture of Compound 6 (2.63 g, 4.0 mmol) and Pd/C (500 mg) in ETOAc(60 mL) in a Parr shaker was stirred under H₂ (65 psi) at roomtemperature overnight. The mixture was filtered though a pad of Celite,washed with MeOH, and concentrated to give a residue which waslyophilized to afford AB4298 (1.2 g, 69% yield). LC-MS: 435 (M+1).

Preparationof16-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-4,7,10-tris(2-methoxyethyl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazahexadecan-1-oicacid (FIG. 27)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added peptoid AB4298 (22.0 mg, 0.05 mmol), FMOC HIPS IndoleCO₂PFP (33.1 mg, 0.05 mmol), NaHCO₃ (4.3 mg, 0.05 mmol), and DMA (0.4mL). The solution was stirred at room temperature for 16 h, acidified topH 2 with 1 M HCl, and extracted with 1×10 mL ETOAc. The organicfraction was washed with a mixture of 1×10 mL 1 M HCl/5 M NaCl (1/1),dried over MgSO₄, filtered, concentrated, adsorbed onto a Biotage SNAPUltra 1 g samplet, and purified on a Biotage SNAP Ultra 10 g cartridgeusing a gradient of 0-10% MeOH in CH₂Cl₂, giving the title compound as awhite solid (28.4 mg, 63% yield). HPLC retention time 12.68 min. MethodA. LRMS (ESI) calcd for C₄₇H₆₁N₇O₁₁ [M−H]⁻: 898.4; found 897.7.

Preparation of (S)-1-((S)-3-maytansinyl)19-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1-indol-1-yl)-7,10,13-tris(2-methoxyethyl)-2,3-dimethyl-4,8,11,14,17-pentaoxo-3,7,10,13,16-pentaazanonadecan-1-oate(FMOC HIPS Indole Glycine Trimethoxypeptoid Beta Alanine Maytansine)(FIG. 27)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added16-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-4,7,10-tris(2-methoxyethyl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazahexadecan-1-oicacid (21.3 mg, 0.02 mmol), deacyl maytansine (15.4 mg, 0.02 mmol), andDMA (0.13 mL). The solution was cooled to 0° C. in an ice, H₂O bathwhereupon 2,4,6-trimethylpyridine (5.7 mg, 6.2 uL, 0.05 mmol) and COMU(13.2 mg, 0.03 mmol) were added. The reaction was allowed to stir at 0°C. for 15 min, then warmed to room temperature and stirred for 3 h. Thereaction mixture was diluted with ETOAc (7 mL), washed with 3×2 mL 0.5 MHCl, 3×2 mL 1.2 M NaHCO₃, and 3×2 mL 5 M NaCl, dried over MgSO₄,filtered, and concentrated to a viscous oil. The product was adsorbedonto a Biotage SNAP Ultra 1 g samplet and purified on a Biotage SNAPUltra 10 g cartridge using a gradient of 2-15% MeOH in CH₂Cl₂, givingthe desired product as a white solid (13.2 mg, 36% yield). HPLCretention time 14.40 min. Method A. LRMS (ESI) calcd for C₇₉H₁₀₃ClN₁₀O₁₉[M+Na]⁺: 1553.7 found 1553.6.

Preparation of (S)-1-((S)-3-maytansinyl)19-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-7,10,13-tris(2-methoxyethyl)-2,3-dimethyl-4,8,11,14,17-pentaoxo-3,7,10,13,16-pentaazanonadecan-1-oate(HIPS Indole Glycine Trimethoxypeptoid Beta Alanine Maytansine) (FIG.27)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC HIPS Indole Glycine Trimethoxypeptoid Beta AlanineMaytansine (13.2 mg, 8.6 umol). Piperidine (14.7 mg, 17.0 uL, 0.17 mmol)in DMA (0.3 mL) was added by syringe. The solution was stirred at roomtemperature for 1 h, adsorbed directly onto a Biotage KP C18 HS 1.2 gsamplet, and purified on a Biotage KP C18 HS 12 g cartridge using agradient of 0-100% CH₃CN in H₂O, giving the title compound as a whitesolid (9.3 mg, 82% yield). HPLC retention time 9.71 min. Method A.

EXAMPLE 15 Method 15—Preparation of (S)-1-((S)-3-maytansinyl)15-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-2,3-dimethyl-4,7,10,13-tetraoxo-3,6,9,12-tetraazapentadecan-1-oate(HIPS Indole Glycine₃ Maytansine)

A reaction scheme for the synthesis of compound HIPS Indole Glycine₃Maytansine is shown in FIG. 28.

Preparation of2-(2-(2-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)acetamido)acetamidotaceticacid (FIG. 28)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added Triglycine (27.4 mg, 0.15 mmol), FMOC HIPS Indole CO₂PFP(46.4 mg, 0.07 mmol), NaHCO₃ (25.9 mg, 0.3 mmol), DMA (0.6 mL), H₂O (0.3mL), and CH₃CN (0.3 mL). The solution was stirred at room temperaturefor 22 h, acidified to pH 2 with 1 M HCl, and extracted with 1×10 mLETOAc. The organic fraction was washed with a mixture of 1×10 mL 1 MHC/5 M NaCl (1/1), dried over MgSO₄, filtered, concentrated, adsorbedonto a Biotage SNAP Ultra 1 g samplet, and purified on a Biotage SNAPUltra 10 g cartridge using a gradient of 0-10% MeOH in CH₂Cl₂, givingthe desired product as a white solid (53.4 mg, 82% yield). HPLCretention time 11.41 min. Method A. LRMS (ESI) calcd for C₃₅H₃₇N₆O₇[M−H]⁻: 653.3; found 652.9.

Preparation of (S)-1-((S)-3-maytansinyl)15-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-2,3-dimethyl-4,7,10,13-tetraoxo-3,6,9,12-tetraazapentadecan-1-oate (FMOCHIPS Indole Glycine₃Maytansine) (FIG. 28)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added2-(2-(2-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)acetamido)acetamido)aceticacid (28.5 mg, 0.03 mmol), deacyl maytansine (28.3 mg, 0.04 mmol), andDMA (0.2 mL). The solution was cooled to 0° C. in an ice, H₂O bathwhereupon 2,4,6-trimethylpyridine (10.5 mg, 11.5 uL, 0.09 mmol) and COMU(20.5 mg, 0.05 mmol) were added. The reaction was allowed to stir at 0°C. for 15 min, then warmed to room temperature and stirred for 16 h. Thereaction mixture was diluted with ETOAc (7 mL), washed with 3×2 mL 0.5 MHCl, 3×2 mL 1.2 M NaHCO₃, and 3×2 mL 5 M NaCl, dried over MgSO₄,filtered, and concentrated to a viscous oil. The product was adsorbedonto a Biotage SNAP Ultra 1 g samplet and purified on a Biotage SNAPUltra 10 g cartridge using a gradient of 2-15% MeOH in CH₂Cl₂, givingthe title compound as a white solid (3.2 mg, 6% yield). HPLC retentiontime 13.63 min. Method A. LRMS (ESI) calcd for C₆₇H₈₀ClN₉O₁₅ [M+Na]⁺:1308.5 found 1308.7.

Preparation of (S)-1-((S)-3-maytansinyl)15-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-2,3-dimethyl-4,7,10,13-tetraoxo-3,6,9,12-tetraazapentadecan-1-oate(HIPS Indole Glycine₃ Maytansine) (FIG. 28)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC HIPS Indole Glycine₃ Maytansine (3.2 mg, 2.5 umol).Piperidine (30.2 mg, 35.0 uL, 0.35 mmol) in DMA (0.2 mL) was added bysyringe. The solution was stirred at room temperature for 1 h andpurified by preparative thin layer chromatography on a Whatman 1000 umpreparative thin layer chromatography plate (SiO₂) using 14% MeOH inCH₂Cl₂, giving the desired product as a white solid (1.8 mg, 68% yield).HPLC retention time 8.81 min. Method A. LRMS (ESI) calcd forC₅₂H₇₀ClN₉O₁₃ [M+H]⁺: 1063.5 found 1064.2.

EXAMPLE 16

Method 16—Preparation of (S)-1-((S)-3-maytansinyl)22-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-2,3-dimethyl-4,14,20-trioxo-7,10-dioxa-3,13,16,19-tetraazadocosan-1-oate(HIPS Indole Aminoethylglycine PEG₂ Maytansine)

Reaction schemes for the synthesis of compound HIPS IndoleAminoethylglycine PEG₂ Maytansine are shown in FIG. 29 and FIG. 30.

Preparation of tert-butyl8-(((9H-fluoren-9-yl)methoxy)carbonyl)-2,2-dimethyl-4,10-dioxo-3,14,17-trioxa-5,8,11-triazaicosan-20-oate(FIG. 29)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added2-((((9H-fluoren-9-yl)methoxy)carbonyl)(2-((tert-butoxycarbonyl)amino)ethyl)amino)aceticacid (90.7 mg, 0.2 mmol), H₂N PEG₂ CO₂tBu (62.0 mg, 0.3 mmol), and DMA(0.8 mL). The solution was cooled to 0° C. in an ice, H₂O bath whereupon2,4,6-trimethylpyridine (49.5 mg, 54.0 uL, 0.4 mmol) and COMU (97.0 mg,0.2 mmol) were added. The reaction was allowed to stir at 0° C. for 15min, then warmed to room temperature and stirred for 1 h. The reactionmixture was diluted with ETOAc (20 mL), washed with 3×7 mL 0.5 M HCl,3×7 mL 1.2 M NaHCO₃, and 3×7 mL 5 M NaCl, dried over MgSO₄, filtered,and concentrated to a viscous oil. The product was adsorbed onto aBiotage SNAP Ultra 3 g samplet and purified on a Biotage SNAP Ultra 25 gcartridge using a gradient of 2-15% MeOH in CH₂Cl₂, giving the titlecompound as a white solid (132.0 mg, 98% yield).

Preparation of3-(((9H-fluoren-9-yl)methoxy)carbonyl)-14-carboxy-5-oxo-9,12-dioxa-3,6-diazatetradecan-1-aminium2,2,2-trifluoroacetate (FIG. 29)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added tert-butyl8-(((9H-fluoren-9-yl)methoxy)carbonyl)-2,2-dimethyl-4,10-dioxo-3,14,17-trioxa-5,8,11-triazaicosan-20-oate(65.5 mg, 0.1 mmol), CH₂Cl₂ (0.5 mL), TFA (0.18 mL), and H₂O (0.02 mL).The reaction was stirred for 2.5 h, dried in vacuo, and azeotroped with3×2 mL Tol, giving the desired product as a waxy residue (55.0 mg, 90%yield). The compound was used in subsequent steps without additionalpurification.

Preparation of7-(((9H-fluoren-9-yl)methoxy)carbonyl)-1-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1-indol-1-yl)-3,9-dioxo-13,16-dioxa-4,7,10-triazanonadecan-19-oicacid (FIG. 29)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added3-(((9H-fluoren-9-yl)methoxy)carbonyl)-14-carboxy-5-oxo-9,12-dioxa-3,6-diazatetradecan-1-aminium2,2,2-trifluoroacetate (55.0 mg, 0.09 mmol), FMOC HIPS Indole CO₂PFP(38.8 mg, 0.06 mmol), NaHCO₃ (30.0 mg, 0.4 mmol), and 1,4 dioxane (0.5mL. The solution was stirred at room temperature for 16 h, acidified topH 2 with 1 M HCl, and extracted with 1×10 mL ETOAc. The organicfraction was washed with a mixture of 1×10 mL 1 M HCl/5 M NaCl (1/1),dried over MgSO₄, filtered, concentrated, adsorbed onto a Biotage SNAPUltra 1 g samplet, and purified on a Biotage SNAP Ultra 10 g cartridgeusing a gradient of 2-8% MeOH in CH₂Cl₂, giving the title compound as awhite solid (28.4 mg, 49% yield). HPLC retention time 15.04 min. MethodA. LRMS (ESI) calcd for C₅₅H₆₀N₆O₁₀ [M+H]⁺: 965; found 965.

Preparation of (S)-1-((S)-3-maytansinyl)16-(((9H-fluoren-9-yl)methoxy)carbonyl)-22-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-2,3-dimethyl-4,14,20-trioxo-7,10-dioxa-3,13,16,19-tetraazadocosan-1-oate(FMOC HIPS Indole Aminoethylglycine (FMOC) PEG₂Maytansine)(FIG. 30)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added7-(((9H-fluoren-9-yl)methoxy)carbonyl)-1-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-3,9-dioxo-13,16-dioxa-4,7,10-triazanonadecan-19-oic acid (30.0 mg, 0.03 mmol), deacyl maytansine (23.7 mg, 0.04mmol), and anhydrous DMF (0.3 mL). The solution was cooled to 0° C. inan ice, H₂O bath whereupon 2,4,6-trimethylpyridine (7.4 mg, 8.1 uL, 0.06mmol) and COMU (15.6 mg, 0.04 mmol) were added. The reaction was allowedto stir at 0° C. for 30 min, then warmed to room temperature and stirredfor 2 h. The reaction mixture was diluted with ETOAc (7 mL), washed with3×2 mL 0.5 M HCl, 3×2 mL 1.2 M NaHCO₃, and 3×2 mL 5 M NaCl, dried overMgSO₄, filtered, and concentrated to a viscous oil. The product wasadsorbed onto a Biotage SNAP Ultra 1 g samplet and purified on a BiotageSNAP Ultra 10 g cartridge using a gradient of 2-7% MeOH in CH₂Cl₂,giving the desired product as a white solid (26.7 mg, 56% yield). HPLCretention time 16.28 min. Method A. LRMS (ESI) calcd for C₈₇H₁₀₂ClN₉O₁₈[M+Na]⁺: 1618.7; found 1619.7.

Preparation of(S)-1-((S)-3-maytansinyl)22-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-2,3-dimethyl-4,14,20-trioxo-7,10-dioxa-3,13,16,19-tetraazadocosan-1-oate(HIPS Indole Aminoethylglycine PEG₂ Maytansine) (FIG. 30)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC HIPS Indole Aminoethylglycine (FMOC) PEG₂ Maytansine(26.6 mg, 17 umol). Piperidine (28.4 mg, 33.0 uL, 0.3 mmol) in DMA (0.16mL) was added by syringe. The solution was stirred at room temperaturefor 1 h, adsorbed onto a Biotage SNAP Ultra 1 g samplet and purified ona Biotage SNAP Ultra 10 g cartridge using 5% MeOH in CH₂Cl₂, giving thetitle compound as a white solid (5.6 mg, 29% yield). HPLC retention time8.10 min. Method A. LRMS (ESI) calcd for C₇₂H₉₂ClN₉O₁₆ [M+H]⁺: 1152.5found 1152.4.

EXAMPLE 17 Method 17—Preparation of (S)-1-((S)-3-maytansinyl)21-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-2,3-dimethyl-4,19-dioxo-14-thioxo-7,10-dioxa-3,13,15,18-tetraazahenicosan-1-oate(HIPS Indole Ethylenediamine Thiourea PEG₂ Maytansine)

Reaction schemes for the synthesis of compound HIPS IndoleEthylenediamine Thiourea PEG₂ Maytansine are shown in FIG. 31 and FIG.32.

Preparation of tert-butyl2,2-dimethyl-4-oxo-9-thioxo-3,13,16-trioxa-5,8,10-triazanonadecan-19-oate(FIG. 31)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added tert-butyl (2-isothiocyanatoethyl)carbamate (129.0 mg, 0.6mmol), H₂N PEG₂ CO₂tBu (166.0 mg, 0.7 mmol), and anhydrous DMF (2.0 mL).The reaction was stirred for 16 h, concentrated in vacuo, adsorbed ontoa Biotage SNAP Ultra 3 g samplet and purified on a Biotage SNAP Ultra 25g cartridge using 30% ETOAc in Hex, giving the desired product as awhite residue (268.0 mg, 96% yield).

Preparation of13-carboxy-4-thioxo-8,11-dioxa-3,5-diazatridecan-1-aminium2,2,2-trifluoroacetate (FIG. 31)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added tert-butyl2,2-dimethyl-4-oxo-9-thioxo-3,13,16-trioxa-5,8,10-triazanonadecan-19-oate(130.0 mg, 0.3 mmol), CH₂Cl₂ (0.5 mL), TFA (0.18 mL), and H₂O (0.02 mL).The reaction was stirred for 2.5 h, dried in vacuo, and azeotroped with3×2 mL Tol, giving the title compound as a waxy residue (116.0 mg, 99%yield). The compound was used in subsequent steps without additionalpurification.

Preparation of1-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-3-oxo-8-thioxo-12,15-dioxa-4,7,9-triazaoctadecan-18-oicacid (FIG. 31)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added 13-carboxy-4-thioxo-8,11-dioxa-3,5-diazatridecan-1-aminium2,2,2-trifluoroacetate (38.4 mg, 0.1 mmol), FMOC HIPS Indole CO₂PFP(38.2 mg, 0.06 mmol), NaHCO₃ (30.0 mg, 0.4 mmol), and 1,4 dioxane (0.5mL. The solution was stirred at room temperature for 16 h, acidified topH 2 with 1 M HCl, and extracted with 1×10 mL ETOAc. The organicfraction was washed with a mixture of 1×10 mL 1 M HCl/5 M NaCl (1/1),dried over MgSO₄, filtered, concentrated, adsorbed onto a Biotage SNAPUltra 1 g samplet, and purified on a Biotage SNAP Ultra 10 g cartridgeusing a gradient of 3-8% MeOH in CH₂Cl₂, giving the desired product as awhite solid (17.6 mg, 40% yield). HPLC retention time 13.02 min. MethodA. LRMS (ESI) calcd for C₃₉H₄₈N₆O₇S [M+H]⁺: 743.3; found 743.1.

Preparation of (S)-1-((S)-3-maytansinyl)21-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-2,3-dimethyl-4,19-dioxo-14-thioxo-7,10-dioxa-3,13,15,18-tetraazahenicosan-1-oate(FMOC HIPS Indole Ethylenediamine Thiourea PEG₂Maytansine) (FIG. 32)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added1-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-3-oxo-8-thioxo-12,15-dioxa-4,7,9-triazaoctadecan-18-oicacid (17.4 mg, 0.02 mmol), HATU (8.9 mg, 0.02 mmol), and DMA (0.1 mL).The clear, colorless solution was stirred at room temperature for 15min. Deacyl maytansine (15.2 mg, 0.02 mmol), DIPEA (6.1 mg, 8.2 uL, 0.05mmol), and anhydrous DMF (0.06 mL) were combined in a separate 1.5 mLmicrocentrifuge tube and added dropwise, slowly, to the stirringsolution. The reaction was allowed to stir at room temperature for 2 h,adsorbed directly onto a Biotage KP C18 HS 1.2 g samplet, and purifiedon a Biotage KP C18 HS 12 g cartridge using a gradient of 0-100% CH₃CNin H₂O, giving the title compound as a white solid (12.7 mg, 39% yield).HPLC retention time 14.71 min. Method A. LRMS (ESI) calcd forC₈₇H₁₀₂ClN₉O₁₈ [M+Na]⁺: 1398.6; found 1398.5.

Preparation of(S)-1-((S)-3-maytansinyl)21-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-2,3-dimethyl-4,19-dioxo-14-thioxo-7,10-dioxa-3,13,15,18-tetraazahenicosan-1-oate(HIPS Indole Ethylenediamine Thiourea PEG₂Maytansine) (FIG. 32)

To a dried 4 mL glass scintillation vial containing a dried flea stirbar was added FMOC HIPS Indole Ethylenediamine Thiourea PEG₂ Maytansine(12.6 mg, 9.2 umol). Piperidine (17.2 mg, 20.0 uL, 0.2 mmol) in DMA (0.1mL) was added by syringe. The solution was stirred at room temperaturefor 1 h, adsorbed directly onto a Biotage KP C18 HS 1.2 g samplet, andpurified on a Biotage KP C18 HS 12 g cartridge using a gradient of0-100% CH₃CN in H₂O, giving the desired product as a white solid (8.6mg, 81% yield). HPLC retention time 9.82 min. Method A. LRMS (ESI) calcdfor C₅₆H₈₀ClN₉O₁₃S [M+H]⁺: 1154.5 found 1154.4.

EXAMPLE 18

Preparation of (2S,15S)-1-((S)-3-maytansinyl)15-(4-aminobutyl)-19-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-2,3-dimethyl-4,14,17-trioxo-7,10-dioxa-3,13,16-triazanonadecan-1-oate(HIPS Indole K (NH₂) PEG₂Maytansine) (Compound 49)

Prepared as in Method 1. HPLC retention time 8.921 min. Method D. LRMS(ESI) calcd for C₅₉H₈₆ClN₉NaO₁₄ ⁺ [M+Na]⁺: 1202.6 found 1202.7.

Preparation of (2S,15S)-1-((S)-3-maytansinyl)19-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-15-(hydroxymethyl)-2,3-dimethyl-4,14,17-trioxo-7,10-dioxa-3,13,16-triazanonadecan-1-oate (HIPS Indole S (OH)PEG₂Maytansine)

Prepared as in Method 3. HPLC retention time 9.147 min. Method A. LRMS(ESI) calcd for C₅₆H₇₉ClN₈NaO₁₅ ⁺ [M+Na]⁺: 1161.5 found 1161.5.

Preparation of (2S,15S)-1-((S)-3-maytansinyl)19-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-2,3-dimethyl-4,14,17-trioxo-15-((phosphonooxy)methyl)-7,10-dioxa-3,13,16-triazanonadecan-1-oate(HIPS Indole S (OPO₃H₂) PEG₂Maytansine)

Prepared as in Method 3. HPLC retention time 8.916 min. Method A. LRMS(ESI) calcd for C₅₆H₇₉ClN₈O₁₈P [M−H]⁻: 1217.5 found 1217.2.

Preparation of (2S,15S)-1-((S)-3-maytansinyl)19-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-2,3-dimethyl-4,14,17-trioxo-15-(4-(Phosphonooxy)benzyl)-7,10-dioxa-3,13,16-triazanonadecan-1-oate(HIPS Indole Y (OPO₃H₂) PEG₂Maytansine)

Prepared as in Method 3. HPLC retention time 9.070 min. Method A. LRMS(ESI) calcd for C₆₂H₈₃ClN₈O₁₈P⁻ [M−H]⁺: 1293.5 found 1293.3.

Preparation of(2S,5S,18R)-2-((S)-3-maytansinyl)-18-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-5,6-dimethyl-4,7,17-trioxo-3,10,13-trioxa-6,16-diazanonadecane-19-sulfonicacid(HIPS Indole C (SO₃H) PEG₂Maytansine)

Prepared as in Method 3. HPLC retention time 9.128 min. Method A. LRMS(ESI) calcd for C₅₆H₇₈ClN₈O₁₇S⁻ [M−H]⁻: 1201.5 found 1201.4.

Preparation of (2S,21S)-1-((S)-3-maytansinyl)25-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-21-(hydroxymethyl)-2,3-dimethyl-4,20,23-trioxo-7,10,13,16-tetraoxa-3,19,22-triazapentacosan-1-oate(HIPS Indole S (OH) PEG₄Maytansine)

Prepared as in Method 3. HPLC retention time 9.228 min. Method A. LRMS(ESI) calcd for C₆₀H₈₇ClN₈NaO₁₇ ⁺ [M+Na]⁺: 1249.6 found 1249.6.

Preparation of (2S,27S)-1-((S)-3-maytansinyl)31-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-27-(hydroxymethyl)-2,3-dimethyl-4,26,29-trioxo-7,10,13,16,19,22-hexaoxa-3,25,28-triazahentriacontan-1-oate(HIPS Indole S (OH) PEG₆ Maytansine)

Prepared as in Method 3. HPLC retention time 9.305 min. Method A. LRMS(ESI) calcd for C₆₄H₉₅ClN₈NaO₁₉ ⁺ [M+Na]⁺: 1337.6 found 1337.6.

Preparation of (S)-1-((S)-3-maytansinyl)2-(6-((S)-2-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-3-hydroxypropanamido)-N-methylhexanamido)propanoate(HIPS Indole S (OH) C₅ Maytansine)

Prepared as in Method 3. HPLC retention time 9.307 min. Method A. LRMS(ESI) calcd for C₅₅H₇₈ClN₈O₁₃ ⁺ [M+H]⁺: 1093.5 found 1093.4.

Preparation of4-((2S,5S)-34-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-5-isopropyl-4,7,29,32-tetraoxo-2-(3-ureidopropyl)-10,13,16,19,22,25-hexaoxa-3,6,28,31-tetraazatetratriacontanamido)benzyl((S)-1-(((S)-1-(((3 R,4S, 5R)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate(HIPS Indole G PEG₆ Val Cit PABC Monomethyl Auristatin E)

Prepared as in Method 7. HPLC retention time 9.832 min. Method A. LRMS(ESI) calcd for C₈₉H₁₄₃N₁₅NaO₂₁ ⁺ [M+Na]⁺: 1781.1 found 1780.9.

Preparation ofN-(5-carboxamidopentyl)-3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamideAlexa Fluor 488 (HIPS Indole Cadaverine Alexa Fluor 488)

Prepared as in Method 4. HPLC retention time 6.448 min. Method A.

Preparation ofN-(5-carboxamidopentyl)-3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamideAlexa Fluor 594 (HIPS Indole Cadaverine Alexa Fluor 594)

Prepared as in Method 4. HPLC retention time 7.666 min. Method A. LRMS(ESI) calcd for C₅₄H₆₂N₇O₁₁S₂ ⁻ [M−H]⁻: 1048.4 found 1048.2.

Preparation ofN-(5-carboxamidopentyl)-3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamideAlexa Fluor 647 (HIPS Indole Cadaverine Alexa Fluor 647)

Prepared as in Method 4. HPLC retention time 5.415 min. Method A.

Preparation ofN-(5-carboxamidopentyl)-3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-ApropanamideATTO 488 (HIPS Indole Cadaverine ATTO 488)

Prepared as in Method 4. HPLC retention time 6.699 min. Method A.

Preparation of(S)-1-carboxamido-13-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-1,12-dioxo-5,8-dioxa-2,11-diazahexadecan-16-oicacid ATTO 647N (HIPS Indole E (CO₂H) PEG₂ NH ATTO 647N)

Prepared as in Method 5. HPLC retention time 11.595 min, 11.896 min(mixture of isomers). Method A.

Preparation of(R)-1-carboxamido-13-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-Apropanamido)-1,12-dioxo-5,8-dioxa-2,11-diazatetradecane-14-sulfonicacid ATTO 647N (HIPS Indole C (SO₃H) PEG₂ NH ATTO 647N)

Prepared as in Method 6. HPLC retention time 12.519 min, 12.884 min(mixture of isomers). Method A.

Preparation of(S)-5-((6-(((S)-1-((S)-3-maytansinyl)-1-oxopropan-2-yl)(methyl)amino)-6-oxohexyl)amino)-4-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-5-oxopentanoicacid (HIPS Indole E (CO₂H) C₅ Maytansine)

Prepared as in Method 1. HPLC retention time 9.361 min. Method A. LRMS(ESI) calcd for C₅₇H₈₀ClN₈O₁₄ ⁺[M+H]⁺: 1135.6 found 1135.4.

Preparation of(S)-5-((4-(((S)-1-((S)-3-maytansinyl)-1-oxopropan-2-yl)(methyl)amino)-4-oxobutyl)amino)-4-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-5-oxopentanoicacid (HIPS Indole E (CO₂H) C₃ Maytansine)

Prepared as in Method 1.HPLC retention time 9.067 min. Method A. LRMS(ESI) calcd for C₅₅H₇₆ClN₈O₁₄ ⁺ [M+H]⁺: 1107.5 found 1107.3.

Preparation of(2S,5S,18S)-2-((S)-3-maytansinyl)-18-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanamido)-5,6-dimethyl-4,7,17-trioxo-3,10,13-trioxa-6,16-diazahenicosan-21-oicacid (HIPS Azaindole E (CO₂H) PEG₂ Maytansine)

Prepared as in Method 1. HPLC retention time 8.534 min. Method A. LRMS(ESI) calcd for C₅₇H₈₀ClN₉NaO₁₆ ⁺ [M+H]⁺: 1204.5 found 1204.5.

Preparation of (S)-1-((S)-3-maytansinyl)16-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)-2,3-dimethyl-4,14-dioxo-7,10-dioxa-3,13-diazahexadecan-1-oate(HIPS Azaindole PEG₂Maytansine)

Prepared as in Method 1. HPLC retention time 8.867 min. Method A. LRMS(ESI) calcd for C₅₂H₇₄ClN₈O₁₃ ⁺ [M+H]⁺: 1053.5 found 1053.3.

Preparation of(S)-1-((S)-3-maytansinyl)2-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)-N-methylpropanamido)propanoate(HIPS Azaindole Maytansine)

Prepared as in Method 1. HPLC retention time 9.198 min. Method A. LRMS(ESI) calcd for C₄₅H₆₁ClN₇O₁₀ ⁺ [M+H]⁺: 894.4 found 894.3.

EXAMPLE 19

General Procedure for Conjugation of HIPS-Linker-Drug to anAldehyde-Tagged Antibody

To conjugate a HIPS-linker-drug to an aldehyde-tagged antibody asdescribed herein, the following general protocol was used. 1.85 mMHIPS-linker-drug (e.g., HIPS-linker-Maytansine) was reacted with 102 μMaldehyde-tagged antibody (e.g., an aldehyde-tagged antibody (dimer) withone aldehyde tag per chain) in PBS with 50 mM sodium citrate pH 5.5,2.5% DMA, and 0.25% Triton X-100 at 37° C. for 16-24 h. After thereaction was complete, unreacted drug was removed using diafiltration.Mono- and di-conjugated species were then purified away fromunconjugated material using a hydrophobic interaction column (GEHealthcare Life Sciences #17-5195-01; Mobile Phase A: 25 mM NaPO₄, 1.0MNH₄ SO₄, pH 7.0; Mobile Phase B: 18.75mM NaPO₄, 25% IPA, pH 7.0). Theenriched sample was put into a final formulation buffer of PBS usingdiafiltration. The final sample was analyzed using hydrophobicinteraction chromatography to determine DAR (Tosoh #14947 ; Mobile PhaseA: 25 mM NaPO₄, 1.5 M NH₄ SO₄, pH 7.0; Mobile Phase B: 18.75 mM NaPO₄,25% IPA, pH 7.0) and size exclusion chromatography determine the levelof aggregation (Tosoh #08541; Mobile Phase A: 25 mM NaPO₄ buffer, 300 mMNaCl, pH 6.8).

Conjugation of HIPS Serine PEG₂Maytansine to an Aldehyde-Tagged Antibody

HIPS Serine PEG₂Maytansine was reacted with an aldehyde-tagged antibodyaccording to the conjugation method described above.

Results are shown in FIGS. 33A-33C, which shows a size exclusionchromatography (SEC) trace (FIG. 33A), a hydrophobic interaction column(HIC) trace (FIG. 33B), and a mass spectrometer (MS) trace (FIG. 33C) ofthe aldehyde-tagged antibody conjugated to HIPS Serine PEG₂Maytansine.The unconjugated, mono-conjugated, and di-conjugated protein conjugateswere observed.

Conjugation of HIPS Phosphoserine PEG₂Maytansine to an Aldehyde-TaggedAntibody

HIPS Phosphoserine PEG₂Maytansine was reacted with an aldehyde-taggedantibody according to the conjugation method described above.

Results are shown in FIGS. 34A-34C, which shows a size exclusionchromatography (SEC) trace (FIG. 34A) and a hydrophobic interactioncolumn (HIC) trace (FIG. 34B), and a mass spectrometer (MS) trace (FIG.34C) of the aldehyde-tagged antibody conjugated to HIPS PhosphoserinePEG₂Maytansine. The unconjugated, mono-conjugated, and di-conjugatedprotein conjugates were observed.

Conjugation of HIPS Cysteic Acid PEG₂Maytansine to an Aldehyde-TaggedAntibody

HIPS Cysteic Acid PEG₂Maytansine was reacted with an aldehyde-taggedantibody according to the conjugation method described above.

Results are shown in FIGS. 35A-35C, which shows a size exclusionchromatography (SEC) trace (FIG. 35A), a hydrophobic interaction column(HIC) trace (FIG. 35B), and a mass spectrometer (MS) trace (FIG. 35C) ofthe aldehyde-tagged antibody conjugated to HIPS HIPS Cysteic AcidPEG₂Maytansine. The unconjugated, mono-conjugated, and di-conjugatedprotein conjugates were observed.

Conjugation of HIPS Glutamic Acid PEG₂Maytansine to an Aldehyde-TaggedAntibody

HIPS Glutamic Acid PEG₂Maytansine was reacted with an aldehyde-taggedantibody according to the conjugation method described above.

Results are shown in FIGS. 36A-36C, which shows a size exclusionchromatography (SEC) trace (FIG. 36A), a hydrophobic interaction column(HIC) trace (FIG. 36B), and a mass spectrometer (MS) trace (FIG. 36C) ofthe aldehyde-tagged antibody conjugated to HIPS Glutamic Acid PEG₂Maytansine. The mono-conjugated and di-conjugated protein conjugateswere observed.

Conjugation of HIPS Asparagine PEG₂ Maytansine to an Aldehyde-TaggedAntibody

HIPS Asparagine PEG₂ Maytansine was reacted with an aldehyde-taggedantibody according to the conjugation method described above.

Results are shown in FIGS. 37A-37B, which shows a size exclusionchromatography (SEC) trace (FIG. 37A) and a hydrophobic interactioncolumn (HIC) trace (FIG. 37B) of the aldehyde-tagged antibody conjugatedto HIPS Asparagine PEG₂ Maytansine. The unconjugated, mono-conjugatedand di-conjugated protein conjugates were observed.

Conjugation of HIPS Phosphotyrosine PEG₂ Maytansine to anAldehyde-Tagged Antibody

HIPS Phosphotyrosine PEG₂ Maytansine was reacted with an aldehyde-taggedantibody according to the conjugation method described above.

Results are shown in FIGS. 38A-38B, which shows a size exclusionchromatography (SEC) trace (FIG. 38A) and a hydrophobic interactioncolumn (HIC) trace (FIG. 38B) of the aldehyde-tagged antibody conjugatedto HIPS Phosphotyrosine PEG₂ Maytansine. The unconjugated,mono-conjugated and di-conjugated protein conjugates were observed.

Conjugation of AzaHIPS Glutamic Acid PEG₂ Maytansine to anAldehyde-Tagged Antibody

AzaHIPS Glutamic Acid PEG₂ Maytansine was reacted with analdehyde-tagged antibody according to the conjugation method describedabove.

Results are shown in FIGS. 39A-39C, which shows a size exclusionchromatography (SEC) trace (FIG. 39A), a hydrophobic interaction column(HIC) trace (FIG. 39B), and a mass spectrometer (MS) trace (FIG. 39C) ofthe aldehyde-tagged antibody conjugated to AzaHIPS Glutamic AcidPEG₂Maytansine. The unconjugated, mono-conjugated and di-conjugatedprotein conjugates were observed.

Conjugation of HIPS Tartaric Acid Maytansine to an Aldehyde-TaggedAntibody

HIPS Tartaric Acid Maytansine was reacted with an aldehyde-taggedantibody according to the conjugation method described above.

Results are shown in FIGS. 40A-40C, which shows a size exclusionchromatography (SEC) trace (FIG. 40A), a hydrophobic interaction column(HIC) trace (FIG. 40B), and a mass spectrometer (MS) trace (FIG. 40C) ofthe aldehyde-tagged antibody conjugated to HIPS Tartaric AcidMaytansine. The unconjugated, mono-conjugated and di-conjugated proteinconjugates were observed.

Conjugation of HIPS Dihydroxy Maytansine to an Aldehyde-Tagged Antibody

HIPS Dihydroxy Maytansine was reacted with an aldehyde-tagged antibodyaccording to the conjugation method described above.

Results are shown in FIGS. 41A-41C, which shows a size exclusionchromatography (SEC) trace (FIG. 41A), a hydrophobic interaction column(HIC) trace (FIG. 41B), and a mass spectrometer (MS) trace (FIG. 41C) ofthe aldehyde-tagged antibody conjugated to HIPS Dihydroxy Maytansine.The mono-conjugated and di-conjugated protein conjugates were observed.

Conjugation of HIPS Glutamic Acid C₃ Maytansine to an Aldehyde-TaggedAntibody

HIPS Glutamic Acid C₃ Maytansine was reacted with an aldehyde-taggedantibody according to the conjugation method described above.

Results are shown in FIGS. 42A-42C, which shows a size exclusionchromatography (SEC) trace (FIG. 42A), a hydrophobic interaction column(HIC) trace (FIG. 42B), and a mass spectrometer (MS) trace (FIG. 42C) ofthe aldehyde-tagged antibody conjugated to HIPS Glutamic Acid C₃Maytansine. The unconjugated, mono-conjugated and di-conjugated proteinconjugates were observed.

Conjugation of HIPS Trimethoxy Maytansine to an Aldehyde-Tagged Antibody

HIPS Trimethoxy Maytansine was reacted with an aldehyde-tagged antibodyaccording to the conjugation method described above.

Results are shown in FIGS. 43A-43C, which shows a size exclusionchromatography (SEC) trace (FIG. 43A), a hydrophobic interaction column(HIC) trace (FIG. 43B), and a mass spectrometer (MS) trace (FIG. 43C) ofthe aldehyde-tagged antibody conjugated to HIPS Trimethoxy Maytansine.The mono-conjugated and di-conjugated protein conjugates were observed.

Conjugation of HIPS Glc NAc PEG₂Ac₃Maytansine to an Aldehyde-TaggedAntibody

HIPS Glc NAc PEG₂ Ac₃ Maytansine was reacted with an aldehyde-taggedantibody according to the conjugation method described above.

Results are shown in FIG. 44, which shows a hydrophobic interactioncolumn (HIC) trace of the aldehyde-tagged antibody conjugated to HIPSGlc NAc PEG₂ Ac₃ Maytansine. The unconjugated, mono-conjugated anddi-conjugated protein conjugates were observed.

Conjugation of HIPS Glc NAc PEG₂ Maytansine to an Aldehyde-TaggedAntibody

HIPS Glc NAc PEG₂ Maytansine was reacted with an aldehyde-taggedantibody according to the conjugation method described above.

Results are shown in FIG. 45, which shows a hydrophobic interactioncolumn (HIC) trace of the aldehyde-tagged antibody conjugated to HIPSGlc NAc PEG₂ Maytansine. The unconjugated, mono-conjugated anddi-conjugated protein conjugates were observed.

Conjugation of HIPS Nit PEG₂ Maytansine to an Aldehyde-Tagged Antibody

HIPS Nit PEG₂ Maytansine was reacted with an aldehyde-tagged antibodyaccording to the conjugation method described above.

Results are shown in FIGS. 46A-46B, which shows a hydrophobicinteraction column (HIC) trace (FIG. 46A) and a mass spectrometer (MS)trace (FIG. 46B) of the aldehyde-tagged antibody conjugated to HIPS NitPEG₂ Maytansine. The unconjugated, mono-conjugated and di-conjugatedprotein conjugates were observed.

Conjugation of HIPS PEG₂ MMAF to an Aldehyde-Tagged Antibody

HIPS PEG₂ MMAF was reacted with an aldehyde-tagged antibody according tothe conjugation method described above.

Results are shown in FIGS. 47A-47B, which shows a hydrophobicinteraction column (HIC) trace (FIG. 47A) and a mass spectrometer (MS)trace (FIG. 47B) of the aldehyde-tagged antibody conjugated to HIPS PEG₂MMAF. The unconjugated, mono-conjugated and di-conjugated proteinconjugates were observed.

Conjugation of HIPS S PEG₄ Maytansine to an Aldehyde-Tagged Antibody

HIPS S PEG₄ Maytansine was reacted with an aldehyde-tagged antibodyaccording to the conjugation method described above.

Results are shown in FIG. 48, which shows a hydrophobic interactioncolumn (HIC) trace of the aldehyde-tagged antibody conjugated to HIPS SPEG₄ Maytansine. The unconjugated, mono-conjugated and di-conjugatedprotein conjugates were observed.

Conjugation of HIPS S PEG₆ Maytansine to an Aldehyde-Tagged Antibody

HIPS S PEG₆ Maytansine was reacted with an aldehyde-tagged antibodyaccording to the conjugation method described above.

Results are shown in FIG. 49, which shows a hydrophobic interactioncolumn (HIC) trace of the aldehyde-tagged antibody conjugated to HIPS SPEG₆ Maytansine. The unconjugated, mono-conjugated and di-conjugatedprotein conjugates were observed.

Conjugation of HIPS S C₅ Maytansine to an Aldehyde-Tagged Antibody

HIPS S C₅ Maytansine was reacted with an aldehyde-tagged antibodyaccording to the conjugation method described above.

Results are shown in FIG. 50, which shows a hydrophobic interactioncolumn (HIC) trace of the aldehyde-tagged antibody conjugated to HIPS SC₅ Maytansine. The unconjugated, mono-conjugated and di-conjugatedprotein conjugates were observed.

Conjugation of HIPS G PEG₆ Maytansine to an Aldehyde-Tagged Antibody

HIPS G PEG₆ Maytansine was reacted with an aldehyde-tagged antibodyaccording to the conjugation method described above.

Results are shown in FIG. 51, which shows a hydrophobic interactioncolumn (HIC) trace of the aldehyde-tagged antibody conjugated to HIPS GPEG₆ Maytansine. The unconjugated, mono-conjugated and di-conjugatedprotein conjugates were observed.

Conjugation of HIPS PEG₆ Val Cit PABC NMC₃ Maytansine to anAldehyde-Tagged Antibody

HIPS PEG₆ Val Cit PABC NMC₃ Maytansine was reacted with analdehyde-tagged antibody according to the conjugation method describedabove.

Results are shown in FIG. 52, which shows a hydrophobic interactioncolumn (HIC) trace of the aldehyde-tagged antibody conjugated to HIPSPEG₆ Val Cit PABC NMC₃ Maytansine. The unconjugated, mono-conjugated anddi-conjugated protein conjugates were observed.

Conjugation of HIPS Gly PEG₆ Val Cit PABC MMAE to an Aldehyde-TaggedAntibody

HIPS Gly PEG₆ Val Cit PABC MMAE was reacted with an aldehyde-taggedantibody according to the conjugation method described above.

Results are shown in FIG. 53, which shows a hydrophobic interactioncolumn (HIC) trace of the aldehyde-tagged antibody conjugated to HIPSGly PEG₆ Val Cit PABC MMAE. The unconjugated, mono-conjugated anddi-conjugated protein conjugates were observed.

Conjugation of HIPS Cysteic Acid Maytansine to an Aldehyde-TaggedAntibody

HIPS Cysteic Acid Maytansine was reacted with an aldehyde-taggedantibody according to the conjugation method described above.

Results are shown in FIG. 54, which shows a hydrophobic interactioncolumn (HIC) trace of the aldehyde-tagged antibody conjugated to HIPSCysteic Acid Maytansine. The unconjugated, mono-conjugated anddi-conjugated protein conjugates were observed.

Conjugation of HIPS Indole E (CO₂H) PEG₂ NH Alexa Fluor 488 to anAldehyde-Tagged Antibody

HIPS Indole E (CO₂H) PEG₂ NH Alexa Fluor 488 was reacted with analdehyde-tagged antibody according to the conjugation method describedabove.

Results are shown in FIG. 55, which shows images of SDS-PAGE gels of thealdehyde-tagged antibody conjugated to HIPS Indole E (CO₂H) PEG₂ NHAlexa Fluor 488. Conjugates to the heavy chain and the light chain ofthe aldehyde-tagged antibody were observed.

Conjugation of HIPS PEG₆ Maytansine to an Aldehyde-Tagged Antibody

HIPS PEG₆ Maytansine was reacted with an aldehyde-tagged antibodyaccording to the conjugation method described above.

Results are shown in FIG. 56, which shows a hydrophobic interactioncolumn (HIC) trace of the aldehyde-tagged antibody conjugated to HIPSPEG₆ Maytansine. The unconjugated, mono-conjugated and di-conjugatedprotein conjugates were observed.

Conjugation of PIPS PEG₂ Maytansine to an Aldehyde-Tagged Antibody

PIPS PEG₂ Maytansine was reacted with an aldehyde-tagged antibodyaccording to the conjugation method described above.

Results are shown in FIG. 57, which shows a hydrophobic interactioncolumn (HIC) trace of the aldehyde-tagged antibody conjugated to PIPSPEG₂ Maytansine. The unconjugated, mono-conjugated and di-conjugatedprotein conjugates were observed.

Conjugation of HIPS Trihydroxy Maytansine to an Aldehyde-Tagged Antibody

HIPS Trihydroxy Maytansine was reacted with an aldehyde-tagged antibodyaccording to the conjugation method described above.

Results are shown in FIG. 58, which shows a hydrophobic interactioncolumn (HIC) trace of the aldehyde-tagged antibody conjugated to HIPSTrihydroxy Maytansine. The unconjugated and conjugated proteinconjugates were observed.

Conjugation of HIPS Lysine PEG₂ Maytansine to an Aldehyde-TaggedAntibody

HIPS Lysine PEG₂ Maytansine was reacted with an aldehyde-tagged antibodyaccording to the conjugation method described above.

Results are shown in FIG. 59, which shows a hydrophobic interactioncolumn (HIC) trace of the aldehyde-tagged antibody conjugated to HIPSLysine PEG₂ Maytansine. The unconjugated and conjugated proteinconjugate was observed.

Conjugation of HIPS-PAPip(PEG2(CO2H)-Maytansine to an Aldehyde-TaggedAntibody

HIPS-PAPip(PEG2(CO2H))-Maytansine was reacted with an aldehyde-taggedantibody according to the conjugation method described above.

Results are shown in FIGS. 66A-66C, which shows a size exclusionchromatography (SEC) trace (FIG. 66A), a hydrophobic interaction column(HIC) trace (FIG. 66B), and a mass spectrometer (MS) trace (FIG. 66C) ofthe aldehyde-tagged antibody conjugated toHIPS-PAPip(PEG2(CO2H))-Maytansine. The unconjugated, mono-conjugated anddi-conjugated protein conjugates were observed.

Conjugation of HIPS-Glutamic Acid-PEG2-Valine-Alanine-PABC-Maytansine toan Aldehyde-Tagged Antibody

HIPS-Glutamic Acid-PEG2-Valine-Alanine-PABC-Maytansine was reacted withan aldehyde-tagged antibody according to the conjugation methoddescribed above.

Results are shown in FIG. 67, which shows a hydrophobic interactioncolumn (HIC) trace of the aldehyde-tagged antibody conjugated toHIPS-Glutamic Acid-PEG2-Valine-Alanine-PABC-Maytansine. Theunconjugated, mono-conjugated and di-conjugated protein conjugates wereobserved.

Conjugation of HIPS-PAPip(PEG2(CO2H))-Valine-Alanine-PABC-MMAD to anAldehyde-Tagged Antibody

HIPS-PAPip(PEG2(CO2H))-Valine-Alanine-PABC-MMAD was reacted with analdehyde-tagged antibody according to the conjugation method describedabove.

Results are shown in FIG. 68, which shows a hydrophobic interactioncolumn (HIC) trace of the aldehyde-tagged antibody conjugated toHIPS-PAPip(PEG2(CO2H))-Valine-Alanine-PABC-MMAD. The unconjugated,mono-conjugated and di-conjugated protein conjugates were observed.

Conjugation of HIPS-PAPip(PEG2(CO2H))-Valine-Citrulline-PABC-MMAD to anAldehyde-Tagged Antibody

HIPS-PAPip(PEG2(CO2H))-Valine-Citrulline-PABC-MMAD was reacted with analdehyde-tagged antibody according to the conjugation method describedabove.

Results are shown in FIGS. 69A-69B, which shows a size exclusionchromatography (SEC) trace (FIG. 69A) and a hydrophobic interactioncolumn (HIC) trace (FIG. 69B) of the aldehyde-tagged antibody conjugatedto HIPS-PAPip(PEG2(CO2H))-Valine-Citrulline-PABC-MMAD. The unconjugated,mono-conjugated and di-conjugated protein conjugates were observed.

Conjugation of AzaHIPS-PAPip(PEG2(CO2H))-Valine-Citrulline-PABC-MMAD toan Aldehyde-Tagged Antibody

AzaHIPS-PAPip(PEG2(CO2H))-Valine-Citrulline-PABC-MMAD was reacted withan aldehyde-tagged antibody according to the conjugation methoddescribed above.

Results are shown in FIGS. 70A-70B, which shows a size exclusionchromatography (SEC) trace (FIG. 70A) and a hydrophobic interactioncolumn (HIC) trace (FIG. 70B) of the aldehyde-tagged antibody conjugatedto AzaHIPS-PAPip(PEG2(CO2H))-Valine-Citrulline-PABC-MMAD. Theunconjugated, mono-conjugated and di-conjugated protein conjugates wereobserved.

Conjugation of HIPS-Glutamic Acid-PEG2-Valine-Citrulline-PABC-Maytansineto an Aldehyde-Tagged Antibody

HIPS-Glutamic Acid-PEG2-Valine-Citrulline-PABC-Maytansine was reactedwith an aldehyde-tagged antibody according to the conjugation methoddescribed above.

Results are shown in FIGS. 71A-71C, which shows a size exclusionchromatography (SEC) trace (FIG. 71A), a hydrophobic interaction column(HIC) trace (FIG. 71B), and a mass spectrometer (MS) trace (FIG. 71C) ofthe aldehyde-tagged antibody conjugated to HIPS-GlutamicAcid-PEG2-Valine-Citrulline-PABC-Maytansine. The unconjugated andmono-conjugated protein conjugates were observed.

Conjugation of HIPS-Asparagine-PEG2-Maytansine to an Aldehyde-TaggedAntibody

HIPS-Asparagine-PEG2-Maytansine was reacted with an aldehyde-taggedantibody according to the conjugation method described above.

Results are shown in FIGS. 72A-72C, which shows a size exclusionchromatography (SEC) trace (FIG. 72A), a hydrophobic interaction column(HIC) trace (FIG. 72B), and a mass spectrometer (MS) trace (FIG. 72C) ofthe aldehyde-tagged antibody conjugated toHIPS-Asparagine-PEG2-Maytansine. The unconjugated, mono-conjugated anddiconjugated protein conjugates were observed.

Conjugation of HIPS-Alanine-PEG2-Maytansine to an Aldehyde-TaggedAntibody

HIPS-Alanine-PEG2-Maytansine was reacted with an aldehyde-taggedantibody according to the conjugation method described above.

Results are shown in FIGS. 73A-73C, which shows a size exclusionchromatography (SEC) trace (FIG. 73A), a hydrophobic interaction column(HIC) trace (FIG. 73B), and a mass spectrometer (MS) trace (FIG. 73C) ofthe aldehyde-tagged antibody conjugated to HIPS-Alanine-PEG2-Maytansine.The unconjugated, mono-conjugated and diconjugated protein conjugateswere observed.

EXAMPLE 20

A reaction scheme for the synthesis of(2S)-1-(((1⁴S,1⁶S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl)oxy)-8-(1-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-2,3-dimethyl-1,4,7-trioxo-11,14-dioxa-3,8-diazaheptadecan-17-oicacid is shown in FIG. 60.

Preparation of tert-butyl 4-oxopiperidine-1-carboxylate (FIG. 60)

To a 100 mL round-bottom flask containing a magnetic stir bar was addedpiperidinone hydrochloride monohydrate (1.53 g, 10 mmol), Boc anhydride(2.39 g, 11 mmol), sodium carbonate (1.22 g, 11.5 mmol), dioxane (10mL), and water (1 mL). The reaction mixture was stirred at roomtemperature for 1 h. The mixture was diluted with water (100 mL) andextracted with ETOAc (3×100 mL). The combined organic layers were washedwith brine, dried over Na₂SO₄, filtered, and concentrated under reducedpressure. The resulting material was dried in vacuo to yield the titlecompound as a white solid (1.74 g, 87% yield).

¹H NMR (CDCl₃) δ 3.73 (t, 4H, J=6.0), 2.46 (t, 4H, J=6.0), 1.51 (s, 9H).

Preparation of tert-butyl4-((2-(2-(3-(tert-butoxy)-3-oxopropoxy)ethoxy)ethyl)amino)piperidine-1-carboxylate(FIG. 60)

To a dried scintillation vial containing a magnetic stir bar was addedtert-butyl 4-oxopiperidine-1-carboxylate (399 mg, 2 mmol),H₂N-PEG₂-COOt-Bu (550 mg, 2.4 mmol), 4 Å molecular sieves (activatedpowder, 200 mg), and 1,2-dichloroethane (5 mL). The mixture was stirredfor 1 h at room temperature. To the reaction mixture was added sodiumtriacetoxyborohydride (845 mg, 4 mmol). The mixture was stirred for 3days at room temperature. The resulting mixture was partitioned betweenETOAc and saturated aqueous NaHCO₃. The organic layer was washed withbrine, dried over Na₂SO₄, filtered, and concentrated under reducedpressure to yield the title compound as a viscous oil (850 mg, >100%yield).

ESI-MS calculated [MH]⁺: 417.3; found 417.2.

Preparation of13-(1-(tert-butoxycarbonyl)piperidin-4-yl)-2,2-dimethyl-4,14-dioxo-3,7,10-trioxa-13-azaheptadecan-17-oicacid (FIG. 60)

To a dried scintillation vial containing a magnetic stir bar was addedtert-butyl4-((2-(2-(3-(tert-butoxy)-3-oxopropoxy)ethoxy)ethyl)amino)piperidine-1-carboxylate(220 mg, 0.5 mmol), succinic anhydride (55 mg, 0.55 mmol),4-(dimethylamino)pyridine (5 mg, 0.04 mmol), and dichloromethane (3 mL).The mixture was stirred for 24 h at room temperature. The reactionmixture was partially purified by flash chromatography (elute 50-100%ETOAc/Hexanes) to yield the title compound as a clear oil (117 mg),which was carried forward without further characterization.

Preparation of 17-(tert-butyl)1-((1⁴S,1⁶S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl)(2S)-8-(1-(tert-butoxycarbonyl)piperidin-4-yl)-2,3-dimethyl-4,7-dioxo-11,14-dioxa-3,8-diazaheptadecanedioate(Boc-PAPip(PEG2(CO2t-Bu)-maytansine) (FIG. 60)

To a dried scintillation vial containing a magnetic stir bar was added13-(1-(tert-butoxycarbonyl)piperidin-4-yl)-2,2-dimethyl-4,14-dioxo-3,7,10-trioxa-13-azaheptadecan-17-oicacid (55 mg, 0.1 mmol), deacyl maytansine (65 mg, 0.1 mmol), HATU (43mg, 0.11 mmol), DMF (1 mL), and dichloromethane (0.5 mL). The mixturewas stirred for 8 h at room temperature. The reaction mixture wasdirectly purified by C18 flash chromatography (elute 5-100% MeCN/water)to yield the title compound as a white film (18 mg, 16% yield).

ESI-MS calculated [MH]⁺: 1148.6; found 1148.7.

Preparation of (2S)-1-(((1⁴S,1⁶S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl)oxy)-2,3-dimethyl-1,4,7-trioxo-8-(piperidin-4-yl)-11,14-dioxa-3,8-diazaheptadecan-17-oicacid (PAPip(PEG2(CO2H))-maytansine) (FIG. 60)

To a dried scintillation vial containing a magnetic stir bar was addedBoc-PAPip(PEG2(CO2t-Bu)-maytansine (31 mg, 0.027 mmol) anddichloromethane (1 mL). The solution was cooled to 0° C. and tin(IV)tetrachloride (1.0 M solution in dichloromethane, 0.3 mL, 0.3 mmol) wasadded. The reaction mixture was stirred for 1 h at 0° C. The reactionmixture was directly purified by C18 flash chromatography (elute 5-100%MeCN/water) to yield the title compound as a white solid (16 mg, 60%yield).

ESI-MS calculated [MH]⁺: 992.5; found 992.6.

Preparation of(2S)-8-(1-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-1-(((1⁴S,1⁶S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl)oxy)-2,3-dimethyl-1,4,7-trioxo-11,14-dioxa-3,8-diazaheptadecan-17-oicacid (Fmoc-HIPS-PAPip(PEG2(CO2H))-maytansine) (FIG. 60)

To a dried scintillation vial containing a magnetic stir bar was addedPAPip(PEG2(CO2H))-maytansine (16 mg, 0.016 mmol),(9H-fluoren-9-yl)methyl1,2-dimethyl-2-((1-(3-oxo-3-(perfluorophenoxy)propyl)-1H-indol-2-yl)methyl)hydrazine-1-carboxylate(13 mg, 0.02 mmol), DIPEA (8 μL, 0.05 mmol), and DMF (1 mL). Thesolution was stirred for 18 h at room temperature. The reaction mixturewas directly purified by C18 flash chromatography (elute 5-100%MeCN/water) to yield the title compound as a white solid (18 mg, 77%yield).

ESI-MS calculated [MH]⁺: 1457.7; found 1457.9.

Preparation of(2S)-1-(((1⁴S,1⁶S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)benzenacyclotetradecaphane-10,12-dien-4-yl)oxy)-8-(1-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-2,3-dimethyl-1,4,7-trioxo-11,14-dioxa-3,8-diazaheptadecan-17-oicacid (FIG. 60)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-HIPS-PAPip(PEG2(CO2H))-maytansine (18 mg, 0.012 mmol), piperidine(20 μL, 0.02 mmol), and DMF (1 mL). The solution was stirred for 20minutes at room temperature. The reaction mixture was directly purifiedby C18 flash chromatography (elute 1-60% MeCN/water) to yield the titlecompound as a white solid (15 mg, 98% yield).

ESI-MS calculated [MH]⁺: 1235.6; found 1236.0.

EXAMPLE 21

A reaction scheme for the synthesis of(2S,5S,18S)-1-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)amino)-18-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-5-isopropyl-2-methyl-1,4,7,17-tetraoxo-10,13-dioxa-3,6,16-triazahenicosan-21-oicacid (HIPS-Glu(OH)-PEG2-Val-Ala-PABC-MMAD) is shown in FIG. 61.

Preparation of4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl((S)-1-(((S)-1-(a3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate(H₂N-Val-Ala-PABC-MMAD)(FIG. 61)

To a dried scintillation vial containing a magnetic stir bar was added(9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)carbamate(102 mg, 0.15 mmol), monomethyl auristatin D (TFA salt, 110 mg, 0.125mmol), HOAt (14 mg, 0.01 mmol), DIPEA (65 μL, 0.37 mmol), and DMA (1mL). The solution was stirred for 20 hours at room temperature. The MMADwas consumed as detected by HPLC. Piperidine (100 μL, 1 mmol) was addedto the reaction mixture and the resulting solution was stirred for anadditional 20 minutes at room temperature. The reaction mixture wasdirectly purified by C18 flash chromatography (elute 5-100% MeCN/water)to yield the title compound as a light yellow solid (101 mg, 75% yield).

ESI-MS calculated [MH]⁺: 1090.6; found 1090.6.

Preparation of tert-butyl(2S,5S,18S)-18-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-1-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,17-tetraoxo-10,13-dioxa-3,6,16-triazahenicosan-21-oate(Fmoc-HIPS-Glu(OtBu)-PEG2-Val-Ala-PABC-MMAD)(FIG. 61)

To a dried scintillation vial containing a magnetic stir bar was added(S)-7-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-2,2-dimethyl-4,8-dioxo-3,12,15-trioxa-9-azaoctadecan-18-oicacid (42 mg, 0.051 mmol), H₂N-Val-Ala-PABC-MMAD (50 mg, 0.046 mmol),HATU (20 mg, 0.053 mmol), DIPEA (16 μL, 0.09 mmol), and DCM (1 mL). Thesolution was stirred for 18 hours at room temperature. An additionalportion of(S)-7-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-2,2-dimethyl-4,8-dioxo-3,12,15-trioxa-9-azaoctadecan-18-oicacid (6 mg, 0.007 mmol), HATU (10 mg, 0.03 mmol), and DIPEA (10 μL, 0.06mmol) were added and the resulting solution was stirred for anadditional 2 hours at room temperature. The reaction mixture wasconcentrated under reduced pressure and subsequently purified by C18flash chromatography (elute 25-100% MeCN/water) to yield the titlecompound as a white solid (82 mg, 94% yield).

ESI-MS calculated [MH]⁺: 1900.0; found 1900.3.

Preparation of (2S, 5S,18S)-18-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-1-((4-((5S,8S, 11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-aS)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(aS)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,17-tetraoxo-10,13-dioxa-3,6,16-triazahenicosan-21-oicacid (Fmoc-HIPS-Glu(OH)-PEG2-Val-Ala-PABC-MMAD)(FIG. 61)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-HIPS-Glu(OtBu)-PEG2-Val-Ala-PABC-MMAD (82 mg, 0.043 mmol) and DCM(0.5 mL). The solution was cooled to 0° C. and tin(IV) tetrachloride wasadded (0.25 mL, 1.0 M in DCM, 0.25 mmol). The resulting mixture wasstirred for 1 hour at 0° C. The reaction mixture was directly purifiedby C18 flash chromatography (elute 5-100% MeCN/water) to yield the titlecompound as a white solid (62 mg, 78% yield).

ESI-MS calculated [MH]⁺: 1844.0; found 1844.2.

Preparation of(2S,5S,185)-1-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)amino)-18-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-Apropanamido)-5-isopropyl-2-methyl-1,4,7,17-tetraoxo-10,13-dioxa-3,6,16-triazahenicosan-21-oicacid (HIPS-Glu(OH)-PEG2-Val-Ala-PABC-MMAD)(FIG. 61)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-HIPS-Glu(OH)-PEG2-Val-Ala-PABC-MMAD (62 mg, 0.034 mmol), piperidine(0.1 mL, 1 mmol) and DMF (0.5 mL). The reaction mixture was stirred for20 minutes at room temperature and then purified by C18 flashchromatography (elute 5-75% MeCN/water) to yield the title compound as awhite solid (25 mg, 46% yield).

ESI-MS calculated [MH]⁺: 1621.9; found 1622.1.

EXAMPLE 22

A reaction scheme for the synthesis of13-(1-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-2,2-dimethyl-4,14-dioxo-3,7,10-trioxa-13-azaheptadecan-17-oicacid (Fmoc-HIPS-PAPip(PEG2(CO2t-Bu))CO2H) is shown in FIG. 62.

Preparation of (9H-fluoren-9-yl)methyl1,2-dimethyl-2-((1-(3-oxo-3-(4-oxopiperidin-1-yl)propyl)-1H-indol-2-yl)methyl)hydrazine-1-carboxylate(Fmoc-HIPS-piperidinone)(FIG. 62)

To a dried scintillation vial containing a magnetic stir bar was added(9H-fluoren-9-yl)methyl1,2-dimethyl-2-((1-(3-oxo-3-(perfluorophenoxy)propyl)-1H-indol-2-yl)methyl)hydrazine-1-carboxylate(700 mg, 1.08 mmol), 4-piperidinone hydrochloride monohydrate (183 mg,1.2 mmol), DIPEA (400 μL, 2.3 mmol), 1,2-dichloroethane (5 mL), and DMF(0.5 mL). The solution was stirred for 2 hours at room temperature. Thereaction mixture was diluted with ETOAc (100 mL) and then washed withHCl (1 M, 3×30 mL), saturated NaHCO₃ (3×30 mL), water (2×30 mL), andbrine (1×30 mL). The organic layer was dried over Na₂SO₄, filtered and,concentrated under reduced pressure, and dried in vacuo to yield thetitle compound as a white solid (355 mg, 58% yield), which was usedwithout further characterization.

Preparation of (9H-fluoren-9-yl)methyl2-((1-(3-(4-((2-(2-(3-(tert-butoxy)-3-oxopropoxy)ethoxy)ethyl)amino)piperidin-1-yl)-3-oxopropyl)-1H-indol-2-yl)methyl)-1,2-dimethylhydrazine-1-carboxylate(Fmoc-HIPS-PAPip(PEG2(CO2t-Bu))NH) (FIG. 62)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-HIPS-piperidinone (355 mg, 0.63 mmol), H₂N-PEG2-COOtBu (175 mg,0.76 mmol), 4 Å molecular sieves (activated powder, 100 mg), and1,2-dichloroethane (2.5 mL). The mixture was stirred for 1 h at roomtemperature. To the reaction mixture was added sodiumtriacetoxyborohydride (314 mg, 1.48 mmol). The mixture was stirred for18 hours at room temperature. The resulting mixture was partitionedbetween ETOAc and saturated aqueous NaHCO₃. The organic layer was washedwith saturated aqueous NaHCO₃ (2×5 mL), and brine (1×5 mL), dried overNa₂SO₄, filtered, and concentrated under reduced pressure to yield thetitle compound as a yellow oil, which was carried forward withoutfurther characterization.

Preparation of13-(1-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-2,2-dimethyl-4,14-dioxo-3,7,10-trioxa-13-azaheptadecan-17-oicacid (Fmoc-HIPS-PAPip(PEG2(CO2t-Bu))CO2H) (FIG. 62)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-HIPS-PAPip(PEG2(CO2t-Bu))NH from the previous step, succinicanhydride (153 mg, 1.53 mmol), 4-(dimethylamino)pyridine (5 mg, 0.04mmol), and dichloromethane (2 mL). The mixture was stirred for 1 h atroom temperature. Methanol (1 mL) was added and the mixture was stirredfor 15 minutes. The reaction mixture was purified by C18 flashchromatography (elute 5-100% MeCN/water with 0.1% acetic acid).Product-containing fractions were concentrated under reduced pressureand then azeotroped with toluene (3×50 mL) to remove residual aceticacid. The title compound was obtained as a foamy white solid (366 mg,38% yield over 3 steps).

ESI-MS calculated [MH]⁺: 882.5; found 882.7.

EXAMPLE 23

A reaction scheme for the synthesis of(2S,5S)-1-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)amino)-11-(1-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-5-isopropyl-2-methyl-1,4,7,10-tetraoxo-14,17-dioxa-3,6,11-triazaicosan-20-oicacid is shown in FIG. 63.

Preparation of tert-butyl (2S, 5S)-11-(1-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyltmethyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-1-((4-((5S,8S,11S,12R)-11-((S)sec-butyl)-5,8-diisopropyl-12-(2-aS)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,10-tetraoxo-14,17-dioxa-3,6,11-triazaicosan-20-oate(Fmoc-HIPS-PAPip(PEG2(CO2t-Bu))-Val-Ala-PABC-MMAD) (FIG. 63)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-HIPS-PAPip(PEG2(CO2t-Bu))CO2H (45 mg, 0.051 mmol),H₂N-Val-Ala-PABC-MMAD (50 mg, 0.046 mmol), HATU (20 mg, 0.053 mmol),DIPEA (16 μL, 0.09 mmol), and DCM (1 mL). The solution was stirred for18 hours at room temperature. The reaction mixture was concentratedunder reduced pressure and subsequently purified by C18 flashchromatography (elute 20-100% MeCN/water) to yield the title compound asa white solid (89 mg, 99% yield).

ESI-MS calculated [MH]⁺: 1954.1; found 1954.3.

Preparation of (2S, 5S)-11-(1-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-1-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-aS)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,10-tetraoxo-14,17-dioxa-3,6,11-triazaicosan-20-oicacid (Fmoc-HIPS-PAPip(PEG2(CO2H))-Val-Ala-PABC-MMAD) (FIG. 63)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-HIPS-PAPip(PEG2(CO2t-Bu))-Val-Ala-PABC-MMAD (89 mg, 0.045 mmol) andDCM (0.5 mL). The solution was cooled to 0° C. and tin(IV) tetrachloridewas added (0.25 mL, 1.0 M in DCM, 0.25 mmol). The resulting mixture wasstirred for 1 hour at 0° C. The reaction mixture was directly purifiedby C18 flash chromatography (elute 10-100% MeCN/water) to yield thetitle compound as a white solid (48 mg, 56% yield).

ESI-MS calculated [MH]⁺: 1898.0; found 1898.2.

Preparation of (2S, 5S)-1-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)amino)-11-(1-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-5-isopropyl-2-methyl-1,4,7,10-tetraoxo-14,17-dioxa-3,6,11-triazaicosan-20-oicacid (FIG. 63)

To a dried scintillation vial containing a magnetic stir bar was added(Fmoc-HIPS-PAPip(PEG2(CO2H))-Val-Ala-PABC-MMAD (48 mg, 0.025 mmol),piperidine (0.1 mL, 1 mmol) and DMF (0.5 mL). The reaction mixture wasstirred for 15 minutes at room temperature and then purified by C18flash chromatography (elute 5-70% MeCN/water) to yield the titlecompound as a white solid (43 mg, >99% yield).

ESI-MS calculated [MH]⁺: 1676.0; found 1676.2.

EXAMPLE 24

A reaction scheme for the synthesis of(6S,9S)-1-amino-6-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-15-(1-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-9-isopropyl-1,8,11,14-tetraoxo-18,21-dioxa-2,7,10,15-tetraazatetracosan-24-oicacid is shown in FIG. 64.

Preparation of4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S, 5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-a(S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate(Fmoc-Val-Cit-PABC-MMAD)(FIG. 64)

To a dried scintillation vial containing a magnetic stir bar was added(9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)carbamate(84 mg, 0.11 mmol), monomethyl auristatin D (TFA salt, 88 mg, 0.1 mmol),HOAt (5 mg, 0.004 mmol), DIPEA (35 μL, 0.2 mmol), and DMA (0.5 mL). Thesolution was stirred for 1 hour at room temperature. To the reactionmixture was added lutidine (35 μL, 0.3 mmol), additional(9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)carbamate(17 mg, 0.02 mmol), and D1PEA (35 μL, 0.2 mmol). The reaction mixturewas heated to 40° C. and stirred for 24 hours. The reaction mixture waspurified by flash chromatography (elute 1-20% MeOH/DCM) to yield thetitle compound as a white solid (107 mg, 76% yield).

ESI-MS calculated [MH]⁺: 1398.8; found 1399.1.

Preparation of4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S, 5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate(H₂N-Val-Cit-PABC-MMAD)(FIG. 64)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-Val-Cit-PABC-MMAD (107 mg, 0.076 mmol), piperidine (0.1 mL, 1 mmol)and DMF (0.5 mL). The reaction mixture was stirred for 20 minutes atroom temperature and then purified by C18 flash chromatography (elute5-100% MeCN/water) to yield the title compound as a white solid (52 mg,58% yield), which was carried forward without further characterization.

Preparation of tert-butyl(6S,9S)-15-(1-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-1-amino-6-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,14-tetraoxo-18,21-dioxa-2,7,10,15-tetraazatetracosan-24-oate(Fmoc-HIPS-PAPip(PEG2(CO2t-Bu))-Val-Cit-PABC-MMAD)(FIG. 64)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-HIPS-PAPip(PEG2(CO2t-Bu))CO2H (28 mg, 0.032 mmol),H₂N-Val-Cit-PABC-MMAD (36 mg, 0.031 mmol), HATU (24 mg, 0.063 mmol),DIPEA (20 μL, 0.12 mmol), DMF (0.5 mL), and DCM (0.6 mL). The solutionwas stirred for 1 hour at room temperature. The reaction mixture waspurified by C18 flash chromatography (elute 25-100% MeCN/water) to yieldthe title compound as a white solid (53 mg, 85% yield).

ESI-MS calculated [MH]⁺: 2040.1; found 2040.5.

Preparation of(6S,9S)-15-(1-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-1-amino-6-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-aS)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,14-tetraoxo-18,21-dioxa-2,7,10,15-tetraazatetracosan-24-oicacid (Fmoc-HIPS-PAPip(PEG2(CO2H))-Val-Cit-PABC-MMAD)(FIG. 64)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-HIPS-PAPip(PEG2(CO2t-Bu))-Val-Cit-PABC-MMAD (53 mg, 0.026 mmol) andDCM (0.8 mL). The solution was cooled to 0° C. and tin(IV) tetrachloridewas added (0.2 mL, 1.0 M in DCM, 0.2 mmol). The resulting mixture wasstirred for 1 hour at 0° C. The reaction mixture was directly purifiedby C18 flash chromatography (elute 5-100% MeCN/water) to yield the titlecompound as a white solid (24 mg, 47% yield).

ESI-MS calculated [MH]⁺: 1984.1; found 1984.3.

Preparation of(6S,9S)-1-amino-6-((4-((5S,8S,11S,12R)-11-((5)-sec-butyl)-5,8-diisopropyl-12-(2-((5)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-15-(1-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-9-isopropyl-1,8,11,14-tetraoxo-18,21-dioxa-2,7,10,15-tetraazatetracosan-24-oicacid (FIG. 64)

To a dried scintillation vial containing a magnetic stir bar was added(Fmoc-HIPS-PAPip(PEG2(CO2H))-Val-Cit-PABC-MMAD (24 mg, 0.012 mmol),piperidine (0.1 mL, 1 mmol) and DMF (0.5 mL). The reaction mixture wasstirred for 15 minutes at room temperature and then purified byC18-flash chromatography (elute 5-80% MeCN/water) to yield the titlecompound as a white solid (15 mg, 70% yield).

ESI-MS calculated [MH]⁺: 1762.0; found 1762.2.

EXAMPLE 25

A reaction scheme for the synthesis of(6S,9S)-1-amino-6-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-15-(1-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanoyl)piperidin-4-yl)-9-isopropyl-1,8,11,14-tetraoxo-18,21-dioxa-2,7,10,15-tetraazatetracosan-24-oicacid is shown in FIG. 65.

Preparation of (9H-fluoren-9-yl)methyl 4-oxopiperidine-1-carboxylate(N-Fmoc-piperidinone)(FIG. 65)

To a 100 mL round-bottom flask containing a magnetic stir bar was addedpiperidinone hydrochloride monohydrate (1.53 g, 10 mmol), Fmoc chloride(2.58 g, 10 mmol), sodium carbonate (3.18 g, 30 mmol), dioxane (20 mL),and water (2 mL). The reaction mixture was stirred at room temperaturefor 1 h. The mixture was diluted with ETOAc (100 mL) and extracted withwater (1×100 mL). The organic layer was dried over Na₂SO₄, filtered, andconcentrated under reduced pressure. The resulting material was dried invacuo to yield the title compound as a white solid (3.05 g, 95% yield).

Preparation of (9H-fluoren-9-yl)methyl4-((2-(2-(3-(tert-butoxy)-3-oxopropoxy)ethoxy)ethyl)amino)piperidine-1-carboxylate(N-Fmoc-piperidine-4-amino-PEG2-COOtBu)(FIG. 65)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-piperidinone (642 mg, 2.0 mmol), H2N-PEG2-COOtBu (560 mg, 2.4mmol), 4 Å molecular sieves (activated powder, 500 mg), and1,2-dichloroethane (5 mL). The mixture was stirred for 1 h at roomtemperature. To the reaction mixture was added sodiumtriacetoxyborohydride (845 mg, 4.0 mmol). The mixture was stirred for 5days at room temperature. The resulting mixture was diluted with ETOAc.The organic layer was washed with saturated NaHCO₃ (1×50 mL), and brine(1×50 mL), dried over Na₂SO₄, filtered, and concentrated under reducedpressure to yield the title compound as an oil, which was carriedforward without further purification.

Preparation of13-(1-(((9H-fluoren-9-yl)methoxy)carbonyl)piperidin-4-yl)-2,2-dimethyl-4,14-dioxo-3,7,10-trioxa-13-azaheptadecan-17-oicacid (Fmoc-PAPip(PEG2(CO2t-Bu))-COOH)(FIG. 65)

To a dried scintillation vial containing a magnetic stir bar was addedN-Fmoc-piperidine-4-amino-PEG2-COOt-Bu from the previous step, succinicanhydride (270 mg, 2.7 mmol), and dichloromethane (5 mL). The mixturewas stirred for 18 hours at room temperature. The reaction mixture waspartitioned between ETOAc and saturated NaHCO₃. The aqueous layer wasextracted with ETOAc (3×). The aqueous layer was acidified with HCl (1M) until the pH 3. The aqueous layer was extracted with DCM (3x). Thecombined organic layers were dried over Na₂SO₄, filtered, andconcentrated under reduced pressure. The reaction mixture was purifiedby C18-flash chromatography (elute 10-100% MeCN/water with 0.1% aceticacid). Product containing fractions were concentrated under reducedpressure and then azeotroped with toluene (3×50 mL) to remove residualacetic acid. The title compound was obtained as a white solid (534 mg,42% yield over 2 steps).

ESI-MS calculated [MH]⁺: 639.3; found 639.2.

Preparation of tert-butyl (6S, 9S)-1-amino-6-((4-((5S,8S,11S,12R)-11((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,14-tetraoxo-15-(piperidin-4-yl)-18,21-dioxa-2,7,10,15-tetraazatetracosan-24-oate(PAPip(PEG2(CO2t-Bu))-Val-Cit-PABC-MMAD)(FIG. 65)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-PAPip(PEG2(CO2t-Bu))-COOH (22 mg, 0.034 mmol),H₂N-Val-Cit-PABC-MMAD (36 mg, 0.031 mmol), HATU (24 mg, 0.063 mmol),DIPEA (20 μL, 0.12 mmol), and DMF (1 mL). The solution was stirred for18 hours at room temperature. Piperidine (0.2 mL, 2 mmol) was added tothe reaction mixture and the resulting solution was stirred for 20minutes at room temperature. The reaction mixture was purified byC18-flash chromatography (elute 5-80% MeCN/water) to yield the titlecompound as a white solid (20 mg, 85% yield).

ESI-MS calculated [MH]⁺: 1574.9; found 1575.1.

Preparation of tert-butyl(6S,9S)-15-(1-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanoyl)piperidin-4-yl)-1-amino-6-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,14-tetraoxo-18,21-dioxa-2,7,10,15-tetraazatetracosan-24-oate(Fmoc -AzaHIPS-PAPip(PEG2(CO2t-Bu))-Val-Cit-PABC-MMAD)(FIG. 65)

To a dried scintillation vial containing a magnetic stir bar was added(PAPip(PEG2(CO2t-Bu))-Val-Cit-PABC-MMAD (20 mg, 0.013 mmol),3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanoicacid (7 mg, 0.014 mmol), PyAOP (8 mg, 0.015 mmol), DIPEA (3 μL, 0.017mmol), and DMF (0.5 mL). The solution was stirred for 2 hours at roomtemperature. The reaction mixture was purified by C18-flashchromatography (elute 5-100% MeCN/water) to yield the title compound asa white solid (20 mg, 77% yield).

ESI-MS calculated [MH]⁺: 2041.1; found 2041.3.

Preparation of(6S,9S)-15-(1-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanoyl)piperidin-4-yl)-1-amino-6-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(aS)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,14-tetraoxo-18,21-dioxa-2,7,10,15-tetraazatetracosan-24-oicacid (Fmoc-AzaHIPS-PAPip(PEG2(CO2H))-Val-Cit-PABC-MMAD)(FIG. 65)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-AzaHIPS-PAPip(PEG2(CO2t-Bu))-Val-Cit-PABC-MMAD (20 mg, 0.01 mmol)and DCM (0.8 mL). The solution was cooled to 0° C. and tin(IV)tetrachloride was added (0.1 mL, 1.0 M in DCM, 0.1 mmol). The resultingmixture was stirred for 1 hour at 0° C. The reaction mixture wasdirectly purified by C18-flash chromatography (elute 10-100% MeCN/water)to yield the title compound as a white solid (14 mg, 72% yield).

ESI-MS calculated [MH]⁺: 1985.1; found 1985.3.

Preparation of (6S,9S)-1-amino-6-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-15-(1-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanoyl)piperidin-4-yl)-9-isopropyl-1,8,11,14-tetraoxo-18,21-dioxa-2,7,10,15-tetraazatetracosan-24-oicacid (FIG. 65)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-AzatlIPS-PAPip(PEG2(CO2H))-Val-Cit-PABC-MMAD (14 mg, 0.007 mmol),piperidine (0.05 mL, 0.5 mmol) and DMF (0.5 mL). The reaction mixturewas stirred for 15 minutes at room temperature and then purified byC18-flash chromatography (elute 5-80% MeCN/water) to yield the titlecompound as a white solid (5 mg, 40% yield).

ESI-MS calculated [MH]⁺: 1763.0; found 1763.2.

EXAMPLE 26

Preparation of tert-butyl(6S,9S,22S)-22-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-1-amino-6-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,21-tetraoxo-14,17-dioxa-2,7,10,20-tetraazapentacosan-25-oate(Fmoc-HIPS-Glu(OtBu)-PEG2-Val-Cit-PABC-MMAD)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-HIPS-Glu(OtBu)-PEG2-COOH (41 mg, 0.042 mmol), H₂N-Val-Cit-PABC-MMAD(52 mg, 0.044 mmol), HATU (20 mg, 0.053 mmol), DIPEA (16 μL, 0.1 mmol),and DCM (1 mL). The solution was stirred for 2 hours at roomtemperature. The reaction mixture was purified by flash chromatography(elute 1-15% MeOH/DCM) to yield the title compound as a white solid (90mg, >99% yield).

ESI-MS calculated [MH]⁺: 1986.1; found 1986.4.

Preparation of (6S,9S,22S)-22-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-1-amino-6-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,21-tetraoxo-14,17-dioxa-2,7,10,20-tetraazapentacosan-25-oicacid (Fmoc-HIPS-Glu(OH)-PEG2-Val-Cit-PABC-MMAD)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-HIPS-Glu(OtBu)-PEG2-Val-Cit-PABC-MMAD (45 mg, 0.023 mmol) and DCM(0.5 mL). The solution was cooled to 0° C. and tin(IV) tetrachloride wasadded (0.2 mL, 1.0 M in DCM, 0.2 mmol). The resulting mixture wasstirred for 1 hour at 0° C. The reaction mixture was directly purifiedby C18 flash chromatography (elute 10-100% MeCN/water) to yield thetitle compound as a white solid (35 mg, 71% yield).

ESI-MS calculated [MH]⁺: 1930.0; found 1930.3.

Preparation of(6S,9S,22S)-1-amino-6-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-22-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-9-isopropyl-1,8,11,21-tetraoxo-14,17-dioxa-2,7,10,20-tetraazapentacosan-25-oicacid (HIPS-Glu(OH)-2PEG-Val-Cit-PABC-MMAD)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-HIPS-Glu-PEG2-Val-Cit-PABC-MMAD (35 mg, 0.018 mmol), piperidine(0.1 mL, 1 mmol) and DMF (0.5 mL). The reaction mixture was stirred for30 minutes at room temperature and then purified by C18 flashchromatography (elute 5-100% MeCN/water) to yield the title compound asa white solid (25 mg, 81% yield).

ESI-MS calculated [MH]⁺: 1708.0; found 1708.1.

EXAMPLE 27

Preparation of tert-butyl(6S,9S,22S)-22-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-1-amino-6-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,21-tetraoxo-14,17-dioxa-2,7,10,20-tetraazapentacosan-25-oate(Fmoc-Glu(OtBu)-PEG2-Val-Cit-PABC-MMAD)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-Glu(OtBu)-PEG2-COOH (25 mg, 0.042 mmol), H₂N-Val-Cit-PABC-MMAD (36mg, 0.031 mmol), HATU (20 mg, 0.053 mmol), D1PEA (20 μL, 0.12 mmol), DCM(0.6 mL), and DMF (0.5 mL). The solution was stirred for 2 hours at roomtemperature. The reaction mixture was purified by C18 flashchromatography (elute 10-100% MeCN/water with 0.1% HOAc) to yield thetitle compound as a white solid (33 mg, 62% yield).

ESI-MS calculated [MH]⁺: 1743.0; found 1743.2.

Preparation of tert-butyl(6S,9S,22S)-1,22-diamino-6-((4-((5S,8S,11S,12R)-11-((5)-sec-butyl)-5,8-diisopropyl-12-(2-((5)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,21-tetraoxo-14,17-dioxa-2,7,10,20-tetraazapentacosan-25-oate(H2N-Glu(OtBu)-2PEG-Val-Cit-PABC-MMAD)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-Glu(OtBu)-PEG2-Val-Cit-PABC-MMAD (33 mg, 0.019 mmol), piperidine(0.1 mL, 1 mmol) and DMF (0.8 mL). The reaction mixture was stirred for15 minutes at room temperature and then purified by C18 flashchromatography (elute 5-100% MeCN/water) to yield the title compound asa white solid (24 mg, 83% yield), which was carried forward withoutfurther characterization.

Preparation of tert-butyl(6S,9S,22S)-22-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanamido)-1-amino-6-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,21-tetraoxo-14,17-dioxa-2,7,10,20-tetraazapentacosan-25-oate(Fmoc-AzaHIPS-Glu(OtBu)-PEG2-Val-Cit-PABC-MMAD)

To a dried scintillation vial containing a magnetic stir bar was added(H2N-Glu(OtBu)-PEG2-Val-Cit-PABC-MMAD (24 mg, 0.016 mmol),3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanoicacid (9 mg, 0.019 mmol), PyAOP (9 mg, 0.017 mmol), DIPEA (3 μL, 0.017mmol), and DMF (0.5 mL). The solution was stirred for 2 hours at roomtemperature. The reaction mixture was purified by C18 flashchromatography (elute 5-100% MeCN/water with 0.1% formic acid) to yieldthe title compound as a white solid (32 mg, >99% yield).

ESI-MS calculated [MH]⁺: 1987.1; found 1987.3.

Preparation of(6S,9S,22S)-22-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanamido)-1-amino-6-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,21-tetraoxo-14,17-dioxa-2,7,10,20-tetraazapentacosan-25-oicacid (Fmoc-AzaHIPS-Glu(OH)-2PEG-Val-Cit-PABC-MMAD)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-AzaHIPS-Glu(OtBu)-2PEG-Val-Cit-PABC-MMAD (32 mg, 0.016 mmol) andDCM (0.5 mL). The solution was cooled to 0° C. and tin(IV) tetrachloridewas added (0.2 mL, 1.0 M in DCM, 0.2 mmol). The resulting mixture wasstirred for 1 hour at 0° C. The reaction mixture was directly purifiedby C18 flash chromatography (elute 10-100% MeCN/water) to yield thetitle compound as a white solid (22 mg, 71% yield).

ESI-MS calculated [MH]⁺: 1931.0; found 1921.2.

Preparation of(6S,9S,22S)-1-amino-6-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-22-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanamido)-9-isopropyl-1,8,11,21-tetraoxo-14,17-dioxa-2,7,10,20-tetraazapentacosan-25-oicacid (AzaHIPS-Glu(OH)-2PEG-Val-Cit-PABC-MMAD)

To a dried scintillation vial containing a magnetic stir bar was addedFmoc-AzaHIPS-Glu-PEG2-Val-Cit-PABC-MMAD (22 mg, 0.011 mmol), piperidine(0.05 mL, 0.5 mmol) and DMF (0.5 mL). The reaction mixture was stirredfor 15 minutes at room temperature and then purified by C18 flashchromatography (elute 5-100% MeCN/water) to yield the title compound asa white solid (15 mg, 77% yield).

ESI-MS calculated [MH]⁺: 1709.0; found 1709.1.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1.-18. (canceled)
 19. A compound of formula (V):

wherein one of Q³ and Q⁴ is —(CH₂)_(m)NR³NHR² and the other is Y⁴; m is0 or 1; R² and R³ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, orR² and R³ are optionally cyclically linked to form a 5 or 6-memberedheterocyclyl; X¹, X², X³ and X⁴ are each independently selected from C,N, O and S, wherein one of X¹, X², X³ and X⁴ is optional; Y¹, Y², Y³ andY⁴ are each independently selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl,wherein Y¹ and Y² or Y² and Y³ are optionally cyclically linked, andwherein when Q⁴ is Y⁴, then Y³ and Y⁴ are optionally cyclically linked;one of R¹⁶, Y¹, Y², Y¹ or Q⁴ is -L-W¹, wherein if Q⁴ is -L-W¹, then Q³is —(CH₂)_(m)NR³NHR² and Y⁴ is absent; and wherein if one of Y¹, Y², Y³or Q⁴ is -L-W¹, then R¹⁶ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; L isa linker comprising-(T¹V¹)_(a)-(T²-V²)_(b)-(T³-V³)_(c)-(T⁴-V⁴)_(d)-(T⁵-V⁵)_(e)-, wherein a,b, c, d and e are each independently 0 or 1, where the sum of a, b, c, dand e is 1 to 5; T¹, T², T³, T⁴ and T⁵ are each independently selectedfrom (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, (EDA)_(w), (PEG)_(n),(AA)_(p), —(CR¹³OH)_(h)—, piperidin-4-amino (P4A),para-amino-benzyloxycarbonyl (PABC), a meta-amino-benzyloxycarbonyl(MABC), a para-amino-benzyloxy (PABO), a meta-amino-benzyloxy (MABO),para-aminobenzyl, an acetal group, a disulfide, a hydrazine, aprotease-cleavable moiety, a glucuronidase cleavable moiety, abeta-lactamase cleavable moiety, and an ester, wherein EDA is anethylene diamine moiety, PEG is a polyethylene glycol or a modifiedpolyethylene glycol, and AA is an amino acid residue; w is an integerfrom 1 to 20; n is an integer from 1 to 30; p is an integer from 1 to20; h is an integer from 1 to 12; V¹, V², V³, V⁴ and V⁵ are eachindependently selected from the group consisting of a covalent bond,—CO—, —NR¹¹—, —CONR¹¹—, —NR¹¹CO—, —C(O)O—, 'OC(O)—, —O—, —S—, —S(O)—,—SO₂—, —SO₂NR¹¹—, —NR¹¹SO₂— and —P(O)OH—; and W¹ is selected from apolypeptide and a chemical entity, and wherein when the sum of a, b, c,d and e is 2 and one of T¹-V¹, T²-V², T³-V³, T⁴-V⁴ or T⁵-V⁵ is (PEG)-CO,then n is not
 6. 20. The compound of claim 19, wherein the compound is acompound of formula (VI):

wherein Q³, Q⁴,X¹, X², X³, X⁴, L, W¹, Y¹, Y² and Y³ are as defined informula (V).
 21. The compound of claim 19, wherein the compound is acompound of formula (VIIa) or (VIIb):

wherein: X¹ and X³ are each independently O, S or NR¹²; in formula(VIIa), one of Q³ and Q⁴ is —(CH₂)_(m)NR³NHR² and the other is Y⁴; informula (VIIb), one of Q³ and R¹², if present, is —(CH₂)_(m)NR³NHR² andthe other is Y³; Y¹, Y², Y³ and Y⁴, if present, are each independentlyselected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, wherein Y¹ and Y² areoptionally cyclically linked, and wherein when Q⁴ is Y⁴, then Y³ and Y⁴are optionally cyclically linked; and m, R² and R³ are as defined informula (V).
 22. The compound of claim 19, wherein Y¹ and Y² or Y³ andY⁴ are cyclically linked to form a fused benzo ring.
 23. The compound ofclaim 19, wherein: T¹ is selected from a (C₁-C₁₂)alkyl and a substituted(C₁-C₁₂)alkyl; T², T³, T⁴ and T⁵ are each independently selected from(EDA)_(w), (PEG)_(n), (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl,(AA)_(p), —(CR¹³OH)_(h)—, piperidin-4-amino, MABC, MABO, PABO, PABC, anacetal group, a disulfide, a hydrazine, a glucuronidase cleavablemoiety, a beta-lactamase cleavable moiety, an ester,(AA)_(p)-MABC-(AA)_(p), (AA)_(p)-MABO-(AA)_(p), (AA)_(p)-PABO-(AA)_(p)and (AA)_(p)-PABC-(AA)_(p); and V¹, V², V³, V⁴ and V⁵ are eachindependently selected from the group consisting of: a covalent bond,—CO—, —NR¹¹—, —CONR¹¹—, —NR¹¹CO—, —C(O)O—, ≧OC(O)—, —O—, —S—, —S(O)—,—SO₂—, —SO₂NR¹¹—, —NR¹¹SO₂—, and —P(O)OH—; wherein: (PEG)_(n) is

where n is an integer from 1 to 30; EDA is an ethylene diamine moietyhaving the following structure:

where q is an integer from 1 to 6 and r is 0 or 1; piperidin-4-amino is

each R¹¹ and R¹² is independently selected from hydrogen, an alkyl, asubstituted alkyl, a polyethylene glycol moiety, an aryl and asubstituted aryl, wherein any two adjacent R¹² groups may be cyclicallylinked to form a piperazinyl ring; and R¹³ is selected from hydrogen, analkyl, a substituted alkyl, an aryl, and a substituted aryl.
 24. Thecompound of claim 19, wherein: a, b, c and d are each 1, and e is 0; ora, b and c are each 1, and d and e are each 0; or a and b are each 1,and c, d and e are each
 0. 25. The compound of claim 19, wherein T¹, T²,T³ and T⁴ and V¹, V², V³ and V⁴ are selected from the following table:T¹ V¹ T² V² T³ V³ T⁴ V⁴ T⁵ V⁵ (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— — —— — — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— — — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) — — — — — — — (C₁-C₁₂)alkyl —CONR¹¹—(PEG)_(n) NR¹¹— — — — — — — (C₁-C₁₂)alkyl —CO— (AA)_(p) NR¹¹— (PEG)_(n)—NR¹¹— — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— — — — — — —(C₁-C₁₂)alkyl —CONR¹¹— (C₁-C₁₂)alkyl —NR¹¹— — — — — — — (C₁-C₁₂)alkyl—CONR¹¹— (PEG)_(n) —CO— (EDA)_(w) — — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w)— — — — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— (CR¹³OH)h —CONR¹¹—(C₁-C₁₂)alkyl —CO— — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (C₁-C₁₂)alkyl—CO— — — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — — — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — MABO — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABO — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABC — — —(C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— (CR¹³OH)h —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (C₁-C₁₂)alkyl —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (C₁-C₁₂)alkyl —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p)- — — — PABC-(AA)_(p) (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p)- —PABC- — (AA)_(p) (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO—(AA)_(p)- — — — PABO (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO—(AA)_(p) — PABO — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —SO₂—(AA)_(p) — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABC- —— — (AA)_(p) (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABC —(AA)_(p) — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— (CR¹³OH)h —CONR¹¹—(PEG)_(n) —CO— — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO—MABC- — — — (AA)_(p)- (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO—MABC — (AA)_(p) — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— MABO — — — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— MABO — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— PABO — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— PABC — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— MABC — (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (CR¹³OH)h —CO— — — — — — — (C₁-C₁₂)alkyl —CONR¹¹—substituted —NR¹¹— (PEG)_(n) —CO— — — — — (C₁-C₁₂)alkyl (C₁-C₁₂)alkyl—SO2— (C₁-C₁₂)alkyl —CO— — — — — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n)—CO— (AA)_(p) — PABC —NR¹¹— — — (C₁-C₁₂)alkyl —CONR¹¹— (C₁-C₁₂)alkyl —(CR¹³OH)h —CONR¹¹— — — — — (C₁-C₁₂)alkyl —CO— P4A —CO— (C₁-C₁₂)alkyl—CO— (AA)_(p) — PABO —CO— (C₁-C₁₂)alkyl —CO— P4A —CO— (C₁-C₁₂)alkyl —CO—(AA)_(p) — PABO — (C₁-C₁₂)alkyl —CO— P4A —CO— (C₁-C₁₂)alkyl —CO—(AA)_(p) — PABC- — (AA)_(p) (C₁-C₁₂)alkyl —CO— P4A —CO— (C₁-C₁₂)alkyl—CO— (AA)_(p) — — — .


26. The compound of claim 19, wherein L is selected from the groupconsisting of the following structures:

wherein: each f is independently 0 or an integer from 1 to 12; each w isindependently 0 or an integer from 1 to 20; each n is independently 0 oran integer from 1 to 30; each p is independently 0 or an integer from 1to 20; each h is independently 0 or an integer from 1 to 12; each R isindependently hydrogen, alkyl, substituted alkyl, a polyethylene glycolmoiety, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; andeach R′ is independently H, a sidechain group of an amino acid, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 27.The compound of claim 19, wherein Q³ is —(CH₂)_(m)NR³NHR² and Q⁴ is Y⁴;or wherein Q⁴ is —(CH₂)_(m)NR³NHR² and Q³ is Y⁴.
 28. The compound ofclaim 19, wherein m is
 1. 29. The compound of claim 19, wherein R² andR³ are each independently selected from alkyl and substituted alkyl. 30.The compound of claim 19, wherein X¹, X², X³ and X⁴ are each C.
 31. Thecompound of claim 19, wherein Y¹, Y², Y³ and Y⁴ are each H.
 32. Thecompound of claim 19, wherein X¹ and X³ are each S and X⁴ is C; orwherein X¹ and X³ are each 0 and X⁴ is C; or wherein X¹ and X³ are eachNR¹² and X⁴ is C.
 33. The compound of claim 19, wherein the compound isa compound of formula (VIII):


34. The compound of claim 19, wherein the compound is a compound offormula (IX):


35. The compound of claim 19, wherein W¹ is a drug or a detectablelabel.
 36. A method of producing a polypeptide conjugate, the methodcomprising: combining in a reaction mixture: a compound of claim 19, anda second compound comprising a reactive aldehyde group or a reactiveketone group, wherein the combining is under reaction conditionssuitable to promote reaction between the compound and the reactivealdehyde group or reactive ketone group of the second compound to formthe conjugate; and isolating the conjugate from the reaction mixture.37. The method of claim 36, wherein W¹ is the chemical entity, and thesecond compound comprises the polypeptide; or wherein W¹ is thepolypeptide, and the second compound comprises the chemical entity.38.-40. (canceled)